Reduced size self-delivering rnai compounds

ABSTRACT

The present invention relates to RNAi constructs with minimal double-stranded regions, and their use in gene silencing. RNAi constructs associated with the invention include a double stranded region of 8-14 nucleotides and a variety of chemical modifications, and are highly effective in gene silencing.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application serial number U.S. 61/192,954, entitled“Chemically Modified Polyucleotides and Methods of Using the Same,”filed on Sep. 22, 2008, U.S. 61/149,946, entitled “Minimum LengthTriggers of RNA Interference,” filed on Feb. 4, 2009, and U.S.61/224,031, entitled “Minimum Length Triggers of RNA Interference,”filed on Jul. 8, 2009, the disclosure of each of which is incorporatedby reference herein in its entirety.

FIELD OF INVENTION

The invention pertains to the field of RNA interference (RNAi). Theinvention more specifically relates to nucleic acid molecules withimproved in vivo delivery properties without the use of a deliveringagent and their use in efficient gene silencing.

BACKGROUND OF INVENTION

Complementary oligonucleotide sequences are promising therapeutic agentsand useful research tools in elucidating gene functions. However, priorart oligonucleotide molecules suffer from several problems that mayimpede their clinical development, and frequently make it difficult toachieve intended efficient inhibition of gene expression (includingprotein synthesis) using such compositions in vivo.

A major problem has been the delivery of these compounds to cells andtissues. Conventional double-stranded RNAi compounds, 19-29 bases long,form a highly negatively-charged rigid helix of approximately 1.5 by10-15 nm in size. This rod type molecule cannot get through thecell-membrane and as a result has very limited efficacy both in vitroand in vivo. As a result, all conventional RNAi compounds require somekind of a delivery vehicle to promote their tissue distribution andcellular uptake. This is considered to be a major limitation of the RNAitechnology.

There have been previous attempts to apply chemical modifications tooligonucleotides to improve their cellular uptake properties. One suchmodification was the attachment of a cholesterol molecule to theoligonucleotide. A first report on this approach was by Letsinger etal., in 1989. Subsequently, ISIS Pharmaceuticals, Inc. (Carlsbad,Calif.) reported on more advanced techniques in attaching thecholesterol molecule to the oligonucleotide (Manoharan, 1992).

With the discovery of siRNAs in the late nineties, similar types ofmodifications were attempted on these molecules to enhance theirdelivery profiles. Cholesterol molecules conjugated to slightly modified(Soutschek, 2004) and heavily modified (Wolfrum, 2007) siRNAs appearedin the literature. Yamada et al., 2008 also reported on the use ofadvanced linker chemistries which further improved cholesterol mediateduptake of siRNAs. In spite of all this effort, the uptake of these typesof compounds appears to be inhibited in the presence of biologicalfluids resulting in highly limited efficacy in gene silencing in vivo,limiting the applicability of these compounds in a clinical setting.

Therefore, it would be of great benefit to improve upon the prior artoligonucleotides by designing oligonucleotides that have improveddelivery properties in vivo and are clinically meaningful.

SUMMARY OF INVENTION

Described herein are asymmetric chemically modified nucleic acidmolecules with minimal double stranded regions, and the use of suchmolecules in gene silencing. RNAi molecules associated with theinvention contain single stranded regions and double stranded regions,and can contain a variety of chemical modifications within both thesingle stranded and double stranded regions of the molecule.Additionally, the RNAi molecules can be attached to a hydrophobicconjugate such as a conventional and advanced sterol-type molecule. Thisnew class of RNAi molecules has superior efficacy both in vitro and invivo than previously described RNAi molecules.

Aspects of the invention relate to asymmetric nucleic acid moleculesincluding a guide strand, with a minimal length of 16 nucleotides, and apassenger strand forming a double stranded nucleic acid, having a doublestranded region and a single stranded region, the double stranded regionhaving 8-15 nucleotides in length, the single stranded region having5-12 nucleotides in length, wherein the passenger strand is linked to alipophilic group, wherein at least 40% of the nucleotides of the doublestranded nucleic acid are modified, and wherein the single strandedregion has at least 2 phosphorothioate modifications. In someembodiments position 1 of the guide strand is 5′ phosphorylated. Incertain embodiments, position 1 of the guide strand is 2′O-methylmodified and 5′ phosphorylated.

Aspects of the invention relate to isolated double stranded nucleic acidmolecules including a longer strand of 15-21 nucleotides in length thathas complementarily to a miRNA sequence, a shorter strand of 8-15nucleotides in length linked at the 3′ end to a lipophilic group,wherein the longer strand and the passenger strand form the doublestranded nucleic acid molecule having a double stranded region and asingle stranded region, wherein the longer strand has a 3′ singlestranded region of 2-13 nucleotides in length, comprising at least twophosphorothioate modification, and at least 50% nucleotides aremodified.

Further aspects of the invention relate to isolated double strandednucleic acid molecules including a guide strand of 17-21 nucleotides inlength that has complementarity to a target gene, a passenger strand of8-16 nucleotides in length linked at the 3′ end to a lipophilic group,wherein the guide strand and the passenger strand form the doublestranded nucleic acid molecule having a double stranded region and asingle stranded region, wherein the guide strand has a 3′ singlestranded region of 2-13 nucleotides in length, each nucleotide withinthe single stranded region having a phosphorothioate modification,wherein the guide strand has a 5′ phosphate modification and wherein atleast 50% of C and U nucleotides in the double stranded region includeat least one 2′ O-methyl modification or 2′-fluoro modification.

In another aspect, the invention is an isolated double stranded nucleicacid molecule having a guide strand of 17-21 nucleotides in length thathas complementarity to a target gene, a passenger strand of 10-16nucleotides in length linked at the 3′ end to a lipophilic group,wherein the guide strand and the passenger strand form the doublestranded nucleic acid molecule having a double stranded region and asingle stranded region, wherein the guide strand has a 3′ singlestranded region of 5-11 nucleotides in length, at least two nucleotidewithin the single stranded region having a phosphorothioatemodification, wherein the guide strand has a 5′ phosphate modificationand wherein at least 50% of C and U nucleotides in the double strandedregion are 2′ O-methyl modification or 2′-fluoro modified.

The invention in another aspect is an isolated double stranded nucleicacid molecule having a guide strand of 17-21 nucleotides in length thathas complementarity to a target gene, a passenger strand of 8-16nucleotides in length linked at the 3′ end to a lipophilic group,wherein the guide strand and the passenger strand form the doublestranded nucleic acid molecule having a double stranded region and asingle stranded region, wherein the guide strand has a 3′ singlestranded region of 6-8 nucleotides in length, each nucleotide within thesingle stranded region having a phosphorothioate modification, whereinthe guide strand has a 5′ phosphate modification, wherein the passengerstrand includes at least two phosphorothioate modifications, wherein atleast 50% of C and U nucleotides in the double stranded region include a2′ O-methyl modification or 2′-fluoro modification, and wherein thedouble stranded nucleic acid molecule has one end that is blunt orincludes a one-two nucleotide overhang.

An isolated double stranded nucleic acid molecule having a guide strandof 17-21 nucleotides in length that has complementarity to a targetgene, a passenger strand of 8-16 nucleotides in length linked at the 3′end to a lipophilic group, wherein the guide strand and the passengerstrand form the double stranded nucleic acid molecule having a doublestranded region and a single stranded region, wherein the guide strandhas a 3′ single stranded region, each nucleotide within the singlestranded region having a phosphorothioate modification, wherein theguide strand has a 5′ phosphate modification, wherein every C and Unucleotide in position 11-18 of the guide strand has a 2′ O-methylmodification, wherein every nucleotide of the passenger strand is 2′O-methyl modified, and wherein the double stranded nucleic acid moleculehas one end that is blunt or includes a one-two nucleotide overhang isprovided in other aspects of the invention.

In another aspect the invention is an isolated double stranded nucleicacid molecule having a guide strand of 17-21 nucleotides in length thathas complementarity to a target gene, a passenger strand of 8-15nucleotides in length linked at the 3′ end to a lipophilic group,wherein the lipophilic group is selected from the group consisting ofcholesterol and a sterol type molecule with C17 polycarbon chain of 5-7or 9-18 carbons in length, wherein the guide strand and the passengerstrand form the double stranded nucleic acid molecule having a doublestranded region and a single stranded region, wherein the guide strandhas a 3′ single stranded region, each nucleotide within the singlestranded region having a phosphorothioate modification, wherein theguide strand has a 5′ phosphate modification, wherein every C and Unucleotide in position 11-18 of the guide strand has a 2′ O-methylmodification, wherein every C and U nucleotide in position 2-10 of theguide strand has a 2′F modification, wherein every nucleotide of thepassenger strand is 2′ O-methyl modified, and wherein the doublestranded nucleic acid molecule has one end that is blunt or includes aone-two nucleotide overhang.

In yet another aspect the invention is an isolated nucleic acid moleculehaving a guide sequence that has complementarity to a target gene, apassenger sequence linked at the 3′ end to a lipophilic group, whereinthe guide sequence and the passenger sequence form a nucleic acidmolecule having a double stranded region and a single stranded region,wherein the guide sequence has a 3′ single stranded region of 2-13nucleotides in length, each nucleotide within the single stranded regionhaving a phosphorothioate modification, wherein the guide sequence has a5′ phosphate modification, wherein at least 50% of C and U nucleotidesin the double stranded region include at least one 2′ O-methylmodification or 2′-fluoro modification, and wherein the double strandednucleic acid molecule has one end that is blunt or includes a one-twonucleotide overhang.

An isolated double stranded nucleic acid molecule having a guide strandand a passenger strand, wherein the region of the molecule that isdouble stranded is from 8-14 nucleotides long, wherein the guide strandcontains a single stranded region that is 4-12 nucleotides long, andwherein the single stranded region of the guide strand contains 2-12phosphorothioate modifications is provided in other aspects of theinvention.

In some embodiments the guide strand contains 6-8 phosphorothioatemodifications. In other embodiments the single stranded region of theguide strand is 6 nucleotides long.

In yet other embodiments the double stranded region is 13 nucleotideslong. Optionally the double stranded nucleic acid molecule has one endthat is blunt or includes a one-two nucleotide overhang.

In another aspect the invention is an isolated double stranded nucleicacid molecule having a guide strand, wherein the guide strand is 16-28nucleotides long and has complementarity to a target gene, wherein the3′ terminal 10 nucleotides of the guide strand include at least twophosphate modifications, and wherein the guide strand has a 5′ phosphatemodification and includes at least one 2′ O-methyl modification or2′-fluoro modification, and a passenger strand, wherein the passengerstrand is 8-14 nucleotides long and has complementarity to the guidestrand, wherein the passenger strand is linked to a lipophilic group,wherein the guide strand and the passenger strand form the doublestranded nucleic acid molecule.

In some embodiments the nucleotide in position one of the guide strandor sequence has a 2′-O-methyl modification. In other embodiments atleast one C or U nucleotide in positions 2-10 of the guide strand orsequence has a 2′-fluoro modification. In yet other embodiments every Cand U nucleotide in positions 2-10 of the guide strand or sequence has a2′-fluoro modification. At least one C or U nucleotide in positions11-18 of the guide strand or sequence may have a 2′-O-methylmodification. In some embodiments every C and U nucleotide in positions11-18 of the guide strand or sequence has a 2′-O-methyl modification.

In yet other embodiments the 3′ terminal 10 nucleotides of the guidestrand include at least four phosphate modifications. Optionally the 3′terminal 10 nucleotides of the guide strand include at least eightphosphate modifications. In some embodiments the guide strand includes4-14 phosphate modifications. In other embodiments the guide strandincludes 4-10 phosphate modifications. In yet other embodiments the 3′terminal 6 nucleotides of the guide strand all include phosphatemodifications. The phosphate modifications may be phosphorothioatemodifications.

In some embodiments every C and U nucleotide on the passenger strand hasa 2′-O-methyl modification. In other embodiments every nucleotide on thepassenger strand has a 2′-O-methyl modification. In an embodiment atleast one nucleotide on the passenger strand is phosphorothioatemodified. At least two nucleotides on the passenger strand arephosphorothioate modified in other embodiments.

The lipophilic molecule may be a sterol, such as cholesterol.

In some embodiments the guide strand is 18-19 nucleotides long. In otherembodiments the passenger strand is 11-13 nucleotides long.

The double stranded nucleic acid molecule has one end that is blunt orincludes a one-two nucleotide overhang in other embodiments.

In other aspects the invention is an isolated double stranded nucleicacid molecule comprising a guide strand and a passenger strand, whereinthe guide strand is from 16-29 nucleotides long and is substantiallycomplementary to a target gene, wherein the passenger strand is from8-14 nucleotides long and has complementarity to the guide strand, andwherein the guide stand has at least two chemical modifications. In someembodiments the at least two chemical modifications include at least twophosphorothioate modifications. In some embodiments the double strandednucleic acid molecule has one end that is blunt or includes a one-twonucleotide overhang.

In some aspects the invention is an isolated double stranded nucleicacid molecule comprising a guide strand and a passenger strand, whereinthe guide strand is from 16-29 nucleotides long and is substantiallycomplementary to a target gene, wherein the passenger strand is from8-14 nucleotides long and has complementarity to the guide strand, andwherein the guide stand has a single stranded 3′ region that is 5nucleotides or longer and a 5′ region that is 1 nucleotide or less. Thesingle stranded region may contain at least 2 phosphorothioatemodifications.

An isolated double stranded nucleic acid molecule having a guide strandand a passenger strand, wherein the guide strand is from 16-29nucleotides long and is substantially complementary to a target gene,wherein the passenger strand is from 8-16 nucleotides long and hascomplementarity to the guide strand, and wherein the guide stand has asingle stranded 3′ region that is 5 nucleotides or longer and apassenger strand has a sterol type molecule with C17 attached chainlonger than 9 is provided in other aspects of the invention.

A duplex polynucleotide is provided in other aspects of the invention.The polynucleotide has a first polynucleotide wherein said firstpolynucleotide is complementary to a second polynucleotide and a targetgene; and a second polynucleotide wherein said second polynucleotide isat least 6 nucleotides shorter than said first polynucleotide, whereinsaid first polynucleotide includes a single stranded region containingmodifications selected from the group consisting of 40-90% hydrophobicbase modifications, 40-90% phosphorothioates, and 40-90% modificationsof the ribose moiety, or any combination thereof.

In other aspects the invention is a duplex polynucleotide having a firstpolynucleotide wherein said first polynucleotide is complementary to asecond polynucleotide and a target gene; and a second polynucleotidewherein said second polynucleotide is at least 6 nucleotides shorterthan said first polynucleotide, wherein the duplex polynucleotideincludes a mismatch between nucleotides 9, 11, 12, 13 or 14 on the firstpolynucleotide and the opposite nucleotide on the second polynucleotide.

In other aspects the invention is a method for inhibiting the expressionof a target gene in a mammalian cell, comprising contacting themammalian cell with an isolated double stranded nucleic acid molecule ofany one of claims 1-41 or a duplex polynucleotide of claim 43 or 44.

A method of inducing RNAi in a subject is provided in other aspects ofthe invention. The method involves administering to a subject aneffective amount for inducing RNAi of an mRNA of a target gene, anisolated double stranded nucleic acid molecule of any one of claims 1-41or a duplex polynucleotide of claim 43 or 44. In other embodiment thesubject is a human. In other embodiments the target gene is PPIB,MAP4K4, or SOD1.

In other aspects an isolated hydrophobic modified polynucleotide havinga polynucleotide, wherein the polynucleotide is double stranded RNA,attached to a hydrophobic molecule, wherein the hydrophobic molecule isattached to a base, a ribose or a backbone of a non-terminal nucleotideand wherein the isolated double stranded nucleic acid molecule comprisesa guide strand and a passenger strand, wherein the guide strand is from16-29 nucleotides long and is substantially complementary to a targetgene, wherein the passenger strand is from 8-14 nucleotides long and hascomplementarity to the guide strand is provided.

In one embodiment the hydrophobic molecule is attached to the guidestrand of the double stranded RNA. In another embodiment the 3′ terminal10 nucleotides of the guide strand include at least two phosphatemodifications, and wherein the guide strand has a 5′ phosphatemodification and includes at least one 2′ O-methyl modification or2′-fluoro modification. In yet another embodiment the hydrophobicmolecule is attached to the passenger strand of the double stranded RNA.

The invention provides an isolated hydrophobic modified polynucleotidehaving a polynucleotide non-covalently complexed to a hydrophobicmolecule, wherein the hydrophobic molecule is a polycationic molecule.In some embodiments the polycationic molecule is selected from the groupconsisting of protamine, arginine rich peptides, and spermine.

In other aspects the invention an isolated hydrophobic modifiedpolynucleotide having a polynucleotide, wherein the polynucleotide isdouble stranded RNA, directly complexed to a hydrophobic moleculewithout a linker, wherein the hydrophobic molecule is not cholesterol.

A composition having a hydrophobic modified polynucleotide, wherein thepolynucleotide is double stranded RNA, attached to a hydrophobicmolecule, wherein the double stranded nucleic acid molecule comprises aguide strand and a passenger strand, wherein the guide strand is from16-29 nucleotides long and is substantially complementary to a targetgene, wherein the passenger strand is from 8-14 nucleotides long and hascomplementarity to the guide strand, wherein position 1 of the guidestrand is 5′ phosphorylated or has a 2′ O-methyl modification, whereinat least 40% of the nucleotides of the double stranded nucleic acid aremodified, and wherein the double stranded nucleic acid molecule has oneend that is blunt or includes a one-two nucleotide overhang; a neutralfatty mixture; and optionally a cargo molecule, wherein the hydrophobicmodified polynucleotide and the neutral fatty mixture forms a micelle isprovided in other aspects of the invention.

In some embodiments the 3′ end of the passenger strand is linked to thehydrophobic molecule. In other embodiments the composition is sterile.In yet other embodiments the neutral fatty mixture comprises a DOPC(dioleoylphosphatidylcholine). In further embodiments the neutral fattymixture comprises a DSPC (distearoylphosphatidylcholine). The neutralfatty mixture further comprises a sterol such as cholesterol in otherembodiments.

In yet other embodiments the composition includes at least 20% DOPC andat least 20% cholesterol. The hydrophobic portion of the hydrophobicmodified polynucleotide is a sterol in other embodiments. The sterol maybe a cholesterol, a cholesteryl or modified cholesteryl residue. Inother embodiments the hydrophobic portion of the hydrophobic modifiedpolynucleotide is selected from the group consisting of bile acids,cholic acid or taurocholic acid, deoxycholate, oleyl litocholic acid,oleoyl cholenic acid, glycolipids, phospholipids, sphingolipids,isoprenoids, vitamins, saturated fatty acids, unsaturated fatty acids,fatty acid esters, triglycerides, pyrenes, porphyrines, Texaphyrine,adamantane, acridines, biotin, coumarin, fluorescein, rhodamine,Texas-Red, digoxygenin, dimethoxytrityl, t-butyldimethylsilyl,t-butyldiphenylsilyl, cyanine dyes (e.g. Cy3 or Cy5), Hoechst 33258 dye,psoralen, and ibuprofen.

In yet other embodiments the hydrophobic portion of the hydrophobicmodified polynucleotide is a polycationic molecule, such as, forinstance, protamine, arginine rich peptides, and/or spermine.

The composition optionally includes a cargo molecule such as a lipid, apeptide, vitamin, and/or a small molecule. In some embodiments the cargomolecule is a commercially available fat emulsions available for avariety of purposes selected from the group consisting of parenteralfeeding. In some embodiments the commercially available fat emulsion isan intralipid or a nutralipid. In other embodiments the cargo moleculeis a fatty acid mixture containing more then 74% of linoleic acid, afatty acid mixture containing at least 6% of cardiolipin, or a fattyacid mixture containing at least 74% of linoleic acid and at least 6% ofcardiolipin. In another embodiment the cargo molecule is a fusogeniclipid, such as for example, DOPE, and preferably is at least 10%fusogenic lipid

In some embodiments the polynucleotide includes chemical modifications.For instance it may be at least 40% modified.

A method of inducing RNAi in a subject is provided in another aspect ofthe invention. The method involves administering to a subject aneffective amount for inducing RNAi of mRNA of a target gene, an isolateddouble stranded nucleic acid molecule or a duplex polynucleotide or acomposition of the invention, wherein the polynucleotide has at least aregion of sequence correspondence to the target gene, wherein the stepof administering is systemic, intravenous, intraperitoneal, intradermal,topical, intranasal, inhalation, oral, intramucosal, local injection,subcutaneous, oral tracheal, or intraocular.

In other embodiment the subject is a human. In other embodiments thetarget gene is PPIB, MAP4K4, or SOD1.

In some aspects the invention is a single-stranded RNA of less than 35nucleotides in length that forms a hairpin structure, said hairpinincludes a double-stranded stem and a single-stranded loop, saiddouble-stranded stem having a 5′-stem sequence having a 5′-end, and a3′-stem sequence having a 3′-end; and said 5′-stem sequence and at leasta portion of said loop form a guide sequence complementary to atranscript of a target gene, wherein said polynucleotide mediatessequence-dependent gene silencing of expression of said target gene,wherein each nucleotide within the single-stranded loop region has aphosphorothioate modification, and wherein at least 50% of C and Unucleotides in the double stranded region include a 2′ O-methylmodification or 2′-fluoro modification. In one embodiment every C and Unucleotide in position 11-18 of the guide sequence has a 2′ O-methylmodification.

A polynucleotide construct is provided in other aspects, thepolynucleotide having two identical single-stranded polynucleotides,each of said single-stranded polynucleotide comprising a 5′-stemsequence having a 5′-end, a 3′-stem sequence having a 3′-end, and alinker sequence linking the 5′-stem sequence and the 3′-stem sequence,wherein: (1) the 5′-stem sequence of a first single-strandedpolynucleotide hybridizes with the 3′-stem sequence of a secondsingle-stranded polynucleotide to form a first double-stranded stemregion; (2) the 5′-stem sequence of the second single-strandedpolynucleotide hybridize with the 3′-stem sequence of the firstsingle-stranded polynucleotide to form a second double-stranded stemregion; and, (3) the linker sequences of the first and the secondsingle-stranded polynucleotides form a loop or bulge connecting saidfirst and said second double-stranded stem regions, wherein the 5′-stemsequence and at least a portion of the linker sequence form a guidesequence complementary to a transcript of a target gene, wherein saidpolynucleotide construct mediates sequence-dependent gene silencing ofexpression of said target gene, wherein each nucleotide within thesingle-stranded loop region has a phosphorothioate modification, andwherein at least 50% of C and U nucleotides in the double strandedregions include a 2′ O-methyl modification or 2′-fluoro modification.

In one embodiment every C and U nucleotide in position 11-18 of theguide sequence has a 2′ O-methyl modification.

In some embodiments, the guide strand is 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, or 29 nucleotides long. In some embodiments, thepassenger strand is 8, 9, 10, 11, 12, 13 or 14 nucleotides long. In someembodiments, the nucleic acid molecule has a thermodynamic stability(ΔG) of less than −20 kkal/mol.

Aspects of the invention relate to nucleic acid molecules that arechemically modified. In some embodiments, the chemical modification isselected from the group consisting of 5′ Phosphate, 2′-O-methyl,2′-O-ethyl, 2′-fluoro, ribothymidine, C-5 propynyl-dC (pdC), C-5propynyl-dU (pdU), C-5 propynyl-C (pC), C-5 propynyl-U (pU), 5-methyl C,5-methyl U, 5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine),5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine, C-5 propynyl-fC (pfC), C-5propynyl-fU (pfU), 5-methyl fC, 5-methyl fU, C-5 propynyl-mC (pmC), C-5propynyl-fU (pmU), 5-methyl mC, 5-methyl mU, LNA (locked nucleic acid),MGB (minor groove binder) and other base modifications which increasebase hydrophobicity. More than one chemical modification may be presentin the same molecule. In some embodiments, chemical modificationincreases stability and/or improves thermodynamic stability (AG). Insome embodiments, at least 90% of CU residues on a nucleic acid moleculeare modified.

In some embodiments, the nucleotide in position one of the guide strandhas a 2′-O-methyl modification and/or a 5′ Phosphate modification. Insome embodiments, at least one C or U nucleotide in positions 2-10 ofthe guide strand has a 2′-fluoro modification. In certain embodiments,every C and U nucleotide in positions 2-10 of the guide strand has a2′-fluoro modification. In some embodiments, at least one C or Unucleotide in positions 11-18 of the guide strand has a 2′-O-methylmodification. In certain embodiments, every C and U nucleotide inpositions 11-18 of the guide strand has a 2′-O-methyl modification. Insome embodiments, every C and U nucleotide on the passenger strand has a2′-O-methyl modification. In certain embodiments, every nucleotide onthe passenger strand has a 2′-O-methyl modification.

In some embodiments, nucleic acid molecules associated with theinvention contain a stretch of at least 4 nucleotides that arephosphorothioate modified. In certain embodiments, the stretch ofnucleotides that are phosphorothioate modified is at least 12nucleotides long. In some embodiments, the stretch of nucleotides thatare phosphorothioate modified is not fully single stranded.

Nucleic acid molecules associated with the invention may be attached toa conjugate. In some embodiments, the conjugate is attached to the guidestrand, while in other embodiments the conjugate is attached to thepassenger strand. In some embodiments, the conjugate is hydrophobic. Insome embodiments, the conjugate is a sterol such as cholesterol. In someembodiments, nucleic acid molecules associated with the invention areblunt-ended.

Aspects of the invention relate to double stranded nucleic acid moleculeincluding a guide strand and a passenger strand, wherein the region ofthe molecule that is double stranded is from 8-14 nucleotides long, andwherein the molecule has a thermodynamic stability (AG) of less than −13kkal/mol.

In some embodiments, the region of the molecule that is double strandedis 8, 9, 10, 11, 12, 13, or 14 nucleotides long. In some embodiments,the molecule has a thermodynamic stability (AG) of less than −20kkal/mol. The nucleic acid molecules, in some embodiments are chemicallymodified. In certain embodiments, the chemical modification is selectedfrom the group consisting of 5′ Phosphate, 2′-O-methyl, 2′-β-ethyl,2′-fluoro, ribothymidine, C-5 propynyl-dC (pdC), C-5 propynyl-dU (pdU),C-5 propynyl-C (pC), C-5 propynyl-U (pU), 5-methyl C, 5-methyl U,5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine),5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine, C-5 propynyl-fC (pfC), C-5propynyl-fU (pfU), 5-methyl fC, 5-methyl fU, C-5 propynyl-mC (pmC), C-5propynyl-fU (pmU), 5-methyl mC, 5-methyl mU, LNA (locked nucleic acid),MGB (minor groove binder) and other base modifications which increasebase hydrophobicity. More than one chemical modification may be presentin the same molecule. In some embodiments, chemical modificationincreases stability and/or improves thermodynamic stability (AG). Insome embodiments, at least 90% of CU residues on a nucleic acid moleculeare modified.

In some embodiments, the nucleotide in position one of the guide strandhas a 2′-O-methyl modification and/or a 5′ Phosphate modification. Insome embodiments, at least one C or U nucleotide in positions 2-10 ofthe guide strand has a 2′-fluoro modification. In certain embodiments,every C and U nucleotide in positions 2-10 of the guide strand has a2′-fluoro modification. In some embodiments, at least one C or Unucleotide in positions 11-18 of the guide strand has a 2′-O-methylmodification. In certain embodiments, every C and U nucleotide inpositions 11-18 of the guide strand has a 2′-O-methyl modification. Insome embodiments, every C and U nucleotide on the passenger strand has a2′-O-methyl modification. In certain embodiments, every nucleotide onthe passenger strand has a 2′-O-methyl modification.

The nucleic acid molecules associated with the invention may contain astretch of at least 4 nucleotides that are phosphorothioate modified. Incertain embodiments, the stretch of nucleotides that arephosphorothioate modified is at least 12 nucleotides long. In someembodiments, the stretch of nucleotides that are phosphorothioatemodified is not fully single stranded. In some embodiments, the nucleicacid molecules are attached to a conjugate. In some embodiments, theconjugate is attached to the guide strand, while in other embodimentsthe conjugate is attached to the passenger strand. In some embodiments,the conjugate is hydrophobic. In some embodiments, the conjugate is asterol such as cholesterol. In some embodiments, nucleic acid moleculesassociated with the invention are blunt-ended. In some embodiments, thenucleic acid molecules are blunt ended at the 5′ end. In certainembodiments, the nucleic acid molecules are blunt ended at the 5′ endwhere the region of complementarity between the two strands of themolecule begins.

Aspects of the invention relate to methods for inhibiting the expressionof a target gene in a mammalian cell. Methods include contacting themammalian cell with an isolated double stranded nucleic acid moleculeincluding a guide strand and a passenger strand, wherein the guidestrand is from 16-29 nucleotides long and has complementarity to atarget gene, wherein the passenger strand is from 8-14 nucleotides longand has complementarity to the guide strand, and wherein the doublestranded nucleic acid molecule has a thermodynamic stability (AG) ofless than −13 kkal/mol.

The cell may be contacted in vivo or in vitro. In some embodiments, theguide strand is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or29 nucleotides long. In some embodiments, the passenger strand is 8, 9,10, 11, 12, 13 or 14 nucleotides long. In some embodiments, the nucleicacid molecule has a thermodynamic stability (AG) of less than −20kkal/mol.

The nucleic acid molecules associated with methods described herein maybe chemically modified. In some embodiments, the chemical modificationis selected from the group consisting of 5′ Phosphate, 2′-O-methyl,2′-O-ethyl, 2′-fluoro, ribothymidine, C-5 propynyl-dC (pdC), C-5propynyl-dU (pdU), C-5 propynyl-C (pC), C-5 propynyl-U (pU), 5-methyl C,5-methyl U, 5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine),5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine, C-5 propynyl-fC (pfC), C-5propynyl-fU (pfU), 5-methyl fC, 5-methyl fU, C-5 propynyl-mC (pmC), C-5propynyl-fU (pmU), 5-methyl mC, 5-methyl mU, LNA (locked nucleic acid),MGB (minor groove binder) and other base modifications which increasebase hydrophobicity. More than one chemical modification may be presentin the same molecule. In some embodiments, chemical modificationincreases stability and/or improves thermodynamic stability (AG). Insome embodiments, at least 90% of CU residues on a nucleic acid moleculeare modified.

In some embodiments, the nucleotide in position one of the guide strandhas a 2′-O-methyl modification and/or a 5′ Phosphate modification. Insome embodiments, at least one C or U nucleotide in positions 2-10 ofthe guide strand has a 2′-fluoro modification. In certain embodiments,every C and U nucleotide in positions 2-10 of the guide strand has a2′-fluoro modification. In some embodiments, at least one C or Unucleotide in positions 11-18 of the guide strand has a 2′-O-methylmodification. In certain embodiments, every C and U nucleotide inpositions 11-18 of the guide strand has a 2′-O-methyl modification. Insome embodiments, every C and U nucleotide on the passenger strand has a2′-O-methyl modification. In certain embodiments, every nucleotide onthe passenger strand has a 2′-O-methyl modification.

In some embodiments, nucleic acid molecules associated with theinvention contain a stretch of at least 4 nucleotides that arephosphorothioate modified. In certain embodiments, the stretch ofnucleotides that are phosphorothioate modified is at least 12nucleotides long. In some embodiments, the stretch of nucleotides thatare phosphorothioate modified is not fully single stranded.

Nucleic acid molecules associated with the invention may be attached toa conjugate. In some embodiments, the conjugate is attached to the guidestrand, while in other embodiments the conjugate is attached to thepassenger strand. In some embodiments, the conjugate is hydrophobic. Insome embodiments, the conjugate is a sterol such as cholesterol. In someembodiments, nucleic acid molecules associated with the invention areblunt-ended.

Methods for inhibiting the expression of a target gene in a mammaliancell described herein include contacting the mammalian cell with anisolated double stranded nucleic acid molecule including a guide strandand a passenger strand, wherein the region of the molecule that isdouble stranded is from 8-14 nucleotides long, and wherein the moleculehas a thermodynamic stability (AG) of less than −13 kkal/mol.

In some embodiments, the region of the molecule that is double strandedis 8, 9, 10, 11, 12, 13, or 14 nucleotides long. In some embodiments,the molecule has a thermodynamic stability (AG) of less than −20kkal/mol. The nucleic acid molecules, in some embodiments are chemicallymodified. In certain embodiments, the chemical modification is selectedfrom the group consisting of 5′ Phosphate, 2′-O-methyl, 2′-β-ethyl,2′-fluoro, ribothymidine, C-5 propynyl-dC (pdC), C-5 propynyl-dU (pdU),C-5 propynyl-C (pC), C-5 propynyl-U (pU), 5-methyl C, 5-methyl U,5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine),5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine, C-5 propynyl-fC (pfC), C-5propynyl-fU (pfU), 5-methyl fC, 5-methyl fU, C-5 propynyl-mC (pmC), C-5propynyl-fU (pmU), 5-methyl mC, 5-methyl mU, LNA (locked nucleic acid),MGB (minor groove binder) and other base modifications which increasebase hydrophobicity. More than one chemical modification may be presentin the same molecule. In some embodiments, chemical modificationincreases stability and/or improves thermodynamic stability (AG). Insome embodiments, at least 90% of CU residues on a nucleic acid moleculeare modified.

In some embodiments, the nucleotide in position one of the guide strandhas a 2′-O-methyl modification and/or a 5′ Phosphate modification. Insome embodiments, at least one C or U nucleotide in positions 2-10 ofthe guide strand has a 2′-fluoro modification. In certain embodiments,every C and U nucleotide in positions 2-10 of the guide strand has a2′-fluoro modification. In some embodiments, at least one C or Unucleotide in positions 11-18 of the guide strand has a 2′-O-methylmodification. In certain embodiments, every C and U nucleotide inpositions 11-18 of the guide strand has a 2′-O-methyl modification. Insome embodiments, every C and U nucleotide on the passenger strand has a2′-O-methyl modification. In certain embodiments, every nucleotide onthe passenger strand has a 2′-O-methyl modification.

The nucleic acid molecules associated with the invention may contain astretch of at least 4 nucleotides that are phosphorothioate modified. Incertain embodiments, the stretch of nucleotides that arephosphorothioate modified is at least 12 nucleotides long. In someembodiments, the stretch of nucleotides that are phosphorothioatemodified is not fully single stranded. In some embodiments, the nucleicacid molecules are attached to a conjugate. In some embodiments, theconjugate is attached to the guide strand, while in other embodimentsthe conjugate is attached to the passenger strand. In some embodiments,the conjugate is hydrophobic. In some embodiments, the conjugate is asterol such as cholesterol. In some embodiments, nucleic acid moleculesassociated with the invention are blunt-ended.

In another embodiment, the invention provides a method for selecting ansiRNA for gene silencing by (a) selecting a target gene, wherein thetarget gene comprises a target sequence; (b) selecting a candidatesiRNA, wherein said candidate siRNA comprises a guide strand of 16-29nucleotide base pairs and a passenger strand of 8-14 nucleotide basepairs that form a duplex comprised of an antisense region and a senseregion and said antisense region of said candidate siRNA is at least 80%complementary to a region of said target sequence; (c) determining athermodynamic stability (AG) of the candidate siRNA; and (e) selectingsaid candidate siRNA as an siRNA for gene silencing, if saidthermodynamic stability is less than −13 kkal/mol.

Aspects of the invention relate to isolated double stranded nucleic acidmolecules including a guide strand and a passenger strand, wherein theguide strand is 18-19 nucleotides long and has complementarity to atarget gene, wherein the passenger strand is 11-13 nucleotides long andhas complementarity to the guide strand, and wherein the double strandednucleic acid molecule has a thermodynamic stability (AG) of less than−13 kkal/mol.

In some embodiments, the nucleotide in position one of the guide strandhas a 2′-O-methyl modification and/or a 5′ Phosphate modification. Insome embodiments, at least one C or U nucleotide in positions 2-10 ofthe guide strand has a 2′-fluoro modification. In certain embodiments,every C and U nucleotide in positions 2-10 of the guide strand has a2′-fluoro modification. In some embodiments, at least one C or Unucleotide in positions 11-18 of the guide strand has a 2′-O-methylmodification. In certain embodiments, every C and U nucleotide inpositions 11-18 of the guide strand has a 2′-O-methyl modification.

In some embodiments, the guide strand contains a stretch of at least 4nucleotides that are phosphorothioate modified. In certain embodiments,the guide strand contains a stretch of at least 8 nucleotides that arephosphorothioate modified. In some embodiments, every C and U nucleotideon the passenger strand has a 2′-O-methyl modification. In certainembodiments, every nucleotide on the passenger strand has a 2′-O-methylmodification. In some embodiments, at least one, or at least twonucleotides on the passenger strand is phosphorothioate modified. Thenucleic acid molecule can be attached to a conjugate on either the guideor passenger strand. In some embodiments, the conjugate is a sterol suchas cholesterol.

Aspects of the invention relate to isolated double stranded nucleic acidmolecules including a guide strand, wherein the guide strand is 16-28nucleotides long and has complementarity to a target gene, wherein the3′ terminal 10 nucleotides of the guide strand include at least twophosphate modifications, and wherein the guide strand includes at leastone 2′ O-methyl modification or 2′-fluoro modification, and a passengerstrand, wherein the passenger strand is 8-28 nucleotides long and hascomplementarity to the guide strand, wherein the passenger strand islinked to a lipophilic group, wherein the guide strand and the passengerstrand form the double stranded nucleic acid molecule.

In some embodiments, the nucleotide in position one of the guide strandhas a 2′-O-methyl modification and/or a 5′ Phosphate modification. Insome embodiments, at least one C or U nucleotide in positions 2-10 ofthe guide strand has a 2′-fluoro modification. In certain embodiments,every C and U nucleotide in positions 2-10 of the guide strand has a2′-fluoro modification. In some embodiments, at least one C or Unucleotide in positions 11-18 of the guide strand has a 2′-O-methylmodification. In certain embodiments, every C and U nucleotide inpositions 11-18 of the guide strand has a 2′-O-methyl modification.

In some embodiments, the 3′ terminal 10 nucleotides of the guide strandinclude at least four, or at least eight phosphate modifications. Incertain embodiments, the guide strand includes 2-14 or 4-10 phosphatemodifications. In some embodiments, the 3′ terminal 6 nucleotides of theguide strand all include phosphate modifications. In certainembodiments, the phosphate modifications are phosphorothioatemodifications.

In some embodiments, every C and U nucleotide on the passenger strandhas a 2′-O-methyl modification. In certain embodiments, every nucleotideon the passenger strand has a 2′-O-methyl modification. In someembodiments, at least one, or at least two nucleotides on the passengerstrand is phosphorothioate modified. In some embodiments, the lipophilicmolecule is a sterol such as cholesterol. In some embodiments, the guidestrand is 18-19 nucleotides long and the passenger strand is 11-13nucleotides long.

Aspects of the invention relate to isolated double stranded nucleic acidmolecules including a guide strand and a passenger strand, wherein theguide strand is from 16-29 nucleotides long and is substantiallycomplementary to a target gene, wherein the passenger strand is from8-14 nucleotides long and has complementarity to the guide strand, andwherein the guide stand has at least two chemical modifications. In someembodiments, the two chemical modifications are phosphorothioatemodifications.

Further aspects of the invention relate to isolated double strandednucleic acid molecule comprising a guide strand and a passenger strand,wherein the guide strand is from 16-29 nucleotides long and issubstantially complementary to a target gene, wherein the passengerstrand is from 8-14 nucleotides long and has complementarity to theguide strand, and wherein the guide stand has a single stranded 3′region that is 5 nucleotides or longer. In some embodiments, the singlestranded region contains at least 2 phosphorothioate modifications.

Further aspects of the invention relate to isolated double strandednucleic acid molecules including a guide strand and a passenger strand,wherein the guide strand is from 18-21 nucleotides long and issubstantially complementary to a target gene, wherein the passengerstrand is from 11-14 nucleotides long and has complementarity to theguide strand, and wherein position one of the guide stand has 2-OMe and5′ phosphate modifications, every C and U in positions 2 to 11 of theguide strand are 2-F modified, every C and U in positions 12-18 of theguide strand are 2′OMe modified, and 80% of Cs and Us on the passengerstrand are 2′OMe modified

Another aspect of the invention relates to isolated double strandednucleic acid molecules including a guide strand and a passenger strand,wherein the guide strand is from 18-21 nucleotides long and issubstantially complementary to a target gene, wherein the passengerstrand is from 11-14 nucleotides long and has complementarity to theguide strand, and wherein the guide stand has 2-OMe and 5′ phosphatemodifications at position 1, every C and U in positions 2 to 11 of theguide strand are 2-F modified, every C and U in positions 12-18 of theguide strand are 2′OMe modified, 80% of Cs and Us on the passengerstrand are 2′OMe and the 3′ end of the passenger strand is attached to aconjugate. In some embodiments the conjugate is selected from sterols,sterol-type molecules, hydrophobic vitamins or fatty acids.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 is a schematic depicting proposed structures of asymmetric doublestranded RNA molecules (adsRNA). Bold lines represent sequences carryingmodification patterns compatible with RISC loading. Striped linesrepresent polynucleotides carrying modifications compatible withpassenger strands. Plain lines represent a single strandedpolynucleotide with modification patterns optimized for cell interactionand uptake. FIG. 1A depicts adsRNA with extended guide or passengerstrands; FIG. 1B depicts adsRNA with length variations of a cellpenetrating polynucleotide; FIG. 1C depicts adsRNA with 3′ and 5′conjugates; FIG. 1D depicts adsRNAs with mismatches.

FIG. 2 is a schematic depicting asymmetric dsRNA molecules withdifferent chemical modification patterns. Several examples of chemicalmodifications that might be used to increase hydrophobicity are shownincluding 4-pyridyl, 2-pyridyl, isobutyl and indolyl based position 5uridine modifications.

FIG. 3 is a schematic depicting the use of dsRNA binding domains,protamine (or other Arg rich peptides), spermidine or similar chemicalstructures to block duplex charge to facilitate cellular entry.

FIG. 4 is a schematic depicting positively charged chemicals that mightbe used for polynucleotide charge blockage.

FIG. 5 is a schematic depicting examples of structural and chemicalcompositions of single stranded RISC entering polynucleotides. Thecombination of one or more modifications including 2′ d, 2′Ome, 2′F,hydrophobic and phosphorothioate modifications can be used to optimizesingle strand entry into the RISC.

FIG. 6 is a schematic depicting examples of structural and chemicalcomposition of RISC substrate inhibitors. Combinations of one or morechemical modifications can be used to mediate efficient uptake andefficient binding to preloaded RISC complex.

FIG. 7 is a schematic depicting structures of polynucleotides withsterol type molecules attached, where R represent a polycarbonic tail of9 carbons or longer. FIG. 7A depicts an adsRNA molecule; FIG. 7B depictsan siRNA molecule of approximately 17-30 bp long; FIG. 7C depicts a RISCentering strand; FIG. 7D depicts a substrate analog strand. Chemicalmodification patterns, as depicted in FIG. 7, can be optimized topromote desired function.

FIG. 8 is a schematic depicting examples of naturally occurringphytosterols with a polycarbon chain that is longer than 8, attached atposition 17. More than 250 different types of phytosterols are known.

FIG. 9 is a schematic depicting examples of sterol-like structures, withvariations in the size of the polycarbon chains attached at position 17.

FIG. 10 presents schematics and graphs demonstrating that the percentageof liver uptake and plasma clearance of lipid emulsions containingsterol type molecules is directly affected by the size of the polycarbonchain attached at position 17. This figure is adapted from Martins etal, Journal of Lipid Research (1998).

FIG. 11 is a schematic depicting micelle formation. FIG. 11A depicts apolynucleotide with a hydrophobic conjugate; FIG. 11B depicts linoleicacid; FIG. 11C depicts a micelle formed from a mixture ofpolynucleotides containing hydrophobic conjugates combined with fattyacids.

FIG. 12 is a schematic depicting how alteration in lipid composition canaffect pharmacokinetic behavior and tissue distribution ofhydrophobically modified and/or hydrophobically conjugatedpolynucleotides. In particular, use of lipid mixtures enriched inlinoleic acid and cardiolipin results in preferential uptake bycardiomyocites.

FIG. 13 is a schematic showing examples of RNAi constructs and controlsused to target MAP4K4 expression. RNAi construct 12083 corresponds toSEQ ID NOs:597 and 598. RNAi construct 12089 corresponds to SEQ IDNO:599.

FIG. 14 is a graph showing MAP4K4 expression following transfection withRNAi constructs associated with the invention. RNAi constructs testedwere: 12083 (Nicked), 12085 (13 nt Duplex), 12089 (No Stem Pairing) and12134 (13 nt miniRNA). Results of transfection were compared to anuntransfected control sample. RNAi construct 12083 corresponds to SEQ IDNOs:597 and 598. RNAi construct 12085 corresponds to SEQ ID NOs:600 and601. RNAi construct 12089 corresponds to SEQ ID NO:599. RNAi construct12134 corresponds to SEQ ID NOs:602 and 603.

FIG. 15 is a graph showing expression of MAP4K4 24 hourspost-transfection with RNAi constructs associated with the invention.RNAi constructs tested were: 11546 (MAP4K4 rxRNA), 12083 (MAP4K4 NickedConstruct), 12134 (12 bp soloRNA) and 12241 (14/3/14 soloRNA). Resultsof transfection were compared to a filler control sample. RNAi construct11546 corresponds to SEQ ID NOs:604 and 605. RNAi construct 12083corresponds to SEQ ID NOs:597 and 598. RNAi construct 12134 correspondsto SEQ ID NOs:602 and 603. RNAi construct 12241 corresponds to SEQ IDNOs:606 and 607.

FIG. 16 presents a graph and several tables comparing parametersassociated with silencing of MAP4K4 expression following transfectionwith RNAi constructs associated with the invention. The rxRNA constructcorresponds to SEQ ID NOs:604 and 605. The 14-3-14 soloRNA constructcorresponds to SEQ ID NOs:606 and 607. The 13/19 duplex (nickedconstruct) corresponds to SEQ ID NOs:597 and 598. The 12-bp soloRNAconstruct corresponds to SEQ ID NOs:602 and 603.

FIG. 17 is a schematic showing examples of RNAi constructs and controlsused to target SOD1 expression. The 12084 RNAi construct corresponds toSEQ ID NOs:612 and 613.

FIG. 18 is a graph showing SOD1 expression following transfection withRNAi constructs associated with the invention. RNAi constructs testedwere: 12084 (Nicked), 12086 (13 nt Duplex), 12090 (No Stem Pairing) and12035 (13 nt MiniRNA). Results of transfection were compared to anuntransfected control sample. The 12084 RNAi construct corresponds toSEQ ID NOs:612 and 613. The 12086 RNAi construct corresponds to SEQ IDNOs:608 and 609. The 12035 RNAi construct corresponds to SEQ ID NOs:610and 611.

FIG. 19 is a graph showing expression of SOD1 24 hours post-transfectionwith RNAi constructs associated with the invention. RNAi constructstested were: 10015 (SOD1 rxRNA) and 12084 (SOD1 Nicked Construct).Results of transfection were compared to a filler control sample. The10015 RNAi construct corresponds to SEQ ID NOs:614 and 615. The 12084RNAi construct corresponds to SEQ ID NOs:612 and 613.

FIG. 20 is a schematic indicating that RNA molecules with doublestranded regions that are less than 10 nucleotides are not cleaved byDicer.

FIG. 21 is a schematic revealing a hypothetical RNAi model for RNAinduced gene silencing.

FIG. 22 is a graph showing chemical optimization of asymmetric RNAicompounds. The presence of chemical modifications, in particular 2′F UC,phosphorothioate modifications on the guide strand, and complete CU2′OMe modification of the passenger strands results in development offunctional compounds. Silencing of MAP4K4 following lipid-mediatedtransfection is shown using RNAi molecules with specific modifications.RNAi molecules tested had sense strands that were 13 nucleotides longand contained the following modifications: unmodified; C and U 2′OMe; Cand U 2′OMe and 3′ Chl; rxRNA 2′OMe pattern; or full 2′OMe, exceptbase 1. Additionally, the guide (anti-sense) strands of the RNAimolecules tested contained the following modifications: unmodified;unmodified with 5′P; C and U 2′F; C and U 2′F with 8 PS 3′ end; andunmodified (17 nt length). Results for rxRNA 12/10 Duplex and negativecontrols are also shown.

FIG. 23 demonstrates that the chemical modifications described hereinsignificantly increase in vitro efficacy in un-assisted delivery of RNAimolecules in HeLa cells. The structure and sequence of the compoundswere not altered; only the chemical modification patterns of themolecules were modified. Compounds lacking 2′ F, 2′O-me,phosphorothioate modification, or cholesterol conjugates were completelyinactive in passive uptake. A combination of all 4 of these types ofmodifications produced the highest levels of activity (compound 12386).

FIG. 24 is a graph showing MAP4K4 expression in Hela cells followingpassive uptake transfection of: NT Accell modified siRNA, MAP4K4 AccellsiRNA, Non-Chl nanoRNA (12379) and sd-nanoRNA (12386).

FIG. 25 is a graph showing expression of MAP4K4 in HeLa cells followingpassive uptake transfection of various concentrations of RNA moleculescontaining the following parameters: Nano Lead with no 3′Chl; Nano Lead;Accell MAP4K4; 21mer GS with 8 PS tail; 21mer GS with 12 PS tail; and25mer GS with 12 PS tail.

FIG. 26 is a graph demonstrating that reduction in oligonucleotidecontent increases the efficacy of unassisted uptake. Similar chemicalmodifications were applied to assymetric compounds, traditional siRNAcompounds and 25 mer RNAi compounds. The assymetric small compoundsdemonstrated the most significant efficacy.

FIG. 27 is a graph demonstrating the importance of phosphorothioatecontent for un-assisted delivery. FIG. 27A demonstrates the results of asystematic screen that revealed that the presence of at least 2-12phosphorothioates in the guide strand significantly improves uptake; insome embodiments, 4-8 phosphorothioate modifications were found to bepreferred. FIG. 27 B reveals that the presence or absence ofphosphorothioate modifications in the sense strand did not alterefficacy.

FIG. 28 is a graph showing expression of MAP4K4 in primary mousehepatocytes following passive uptake transfection of: AccellMedia-Ctrl-UTC; MM APOB Alnylam; Active APOB Alnylam; nanoRNA withoutchl; nanoRNA MAP4K4; Mouse MAP4K4 Accell Smartpool; DY547 AccellControl; Luc Ctrl rxRNA with Dy547; MAP4K4 rxRNA with DY547; and ASStrand Alone (nano).

FIG. 29 is a graph showing expression of ApoB in mouse primaryhepatocytes following passive uptake transfection of: AccellMedia-Ctrl-UTC; MM APOB Alnylam; Active APOB Alnylam; nanoRNA withoutchl; nanoRNA MAP4K4; Mouse MAP4K4 Accell Smartpool; DY547 AccellControl; Luc Ctrl rxRNA with Dy547; MAP4K4 rxRNA with DY547; and ASStrand Alone (nano).

FIG. 30 is a graph showing expression of MAP4K4 in primary humanhepatocytes following passive uptake transfection of: 11550 MAP4K4rxRNA; 12544 mM MAP4K4 nanoRNA; 12539 Active MAP4K4 nanoRNA; AccellMedia; and UTC.

FIG. 31 is a graph showing ApoB expression in primary human hepatoctyesfollowing passive uptake transfection of: 12505 Active ApoB chol-siRNA;12506 mM ApoB chol-siRNA; Accell Media; and UTC.

FIG. 32 is an image depicting localization of sd-rxRNA^(nano)localization.

FIG. 33 is an image depicting localization of Chol-siRNA (Alnylam).

FIG. 34 is a schematic of 1^(st) generation (G1) sd-rxRNA^(nano)molecules associated with the invention indicating regions that aretargeted for modification, and functions associated with differentregions of the molecules.

FIG. 35 depicts modification patterns that were screened foroptimization of sd-rxRNA' (G1). The modifications that were screenedincluded, on the guide strand, lengths of 19, 21 and 25 nucleotides,phosphorothioate modifications of 0-18 nucleotides, and replacement of2′F modifications with 2′OMe, 5 Methyl C and/or ribo Thymidinemodifications. Modifications on the sense strand that were screenedincluded nucleotide lengths of 11, 13 and 19 nucleotides,phosphorothiote modifications of 0-4 nucleotides and 2′OMemodifications.

FIG. 36 is a schematic depicting modifications of sd-rxRNA^(nano) thatwere screened for optimization.

FIG. 37 is a graph showing percent MAP4K4 expression in Hek293 cellsfollowing transfection of: Risc Free siRNA; rxRNA; Nano (unmodified); GSalone; Nano Lead (no Chl); Nano (GS: (3) 2′OMe at positions 1, 18, and19, 8 PS, 19 nt); Nano (GS: (3) 2′OMe at positions 1, 18, and 19, 8 PS,21 nt); Nano (GS: (3) 2′OMe at positions 1, 18, and 19, 12 PS, 21 nt);and Nano (GS: (3) 2′OMe at positions 1, 18, and 19, 12 PS, 25 nt);

FIG. 38 is a graph showing percent MAP4K4 expression in HeLa cellsfollowing passive uptake transfection of: GS alone; Nano Lead; Nano (GS:(3) 2′OMe at positions 1, 18, and 19, 8 PS, 19 nt); Nano (GS: (3) 2′OMeat positions 1, 18, and 19, 8 PS, 21 nt); Nano (GS: (3) 2′OMe atpositions 1, 18, and 19, 12 PS, 21 nt); Nano (GS: (3) 2′OMe at positions1, 18, and 19, 12 PS, 25 nt).

FIG. 39 is a graph showing percent MAP4K4 expression in Hek293 cellsfollowing lipid mediated transfection of: Guide Strand alone (GS: 8 PS,19 nt); Guide Strand alone (GS: 18 PS, 19 nt); Nano (GS: no PS, 19 nt);Nano (GS: 2 PS, 19 nt); Nano (GS: 4 PS, 19 nt); Nano (GS: 6 PS, 19 nt);Nano Lead (GS: 8 PS, 19 nt); Nano (GS: 10 PS, 19 nt); Nano (GS: 12 PS,19 nt); and Nano (GS: 18 PS, 19 nt).

FIG. 40 is a graph showing percent MAP4K4 expression in Hek293 cellsfollowing lipid mediated transfection of: Guide Strand alone (GS: 8 PS,19 nt); Guide Strand alone (GS: 18 PS, 19 nt); Nano (GS: no PS, 19 nt);Nano (GS: 2 PS, 19 nt); Nano (GS: 4 PS, 19 nt); Nano (GS: 6 PS, 19 nt);Nano Lead (GS: 8 PS, 19 nt); Nano (GS: 10 PS, 19 nt); Nano (GS: 12 PS,19 nt); and Nano (GS: 18 PS, 19 nt).

FIG. 41 is a graph showing percent MAP4K4 expression in HeLa cellsfollowing passive uptake transfection of: Nano Lead (no Chl); GuideStrand alone (18 PS); Nano (GS: 0 PS, 19 nt); Nano (GS: 2 PS, 19 nt);Nano (GS: 4 PS, 19 nt); Nano (GS: 6 PS, 19 nt); Nano Lead (GS: 8 PS, 19nt); Nano (GS: 10 PS, 19 nt); Nano (GS: 12 PS, 19 nt); and Nano (GS: 18PS, 19 nt).

FIG. 42 is a graph showing percent MAP4K4 expression in HeLa cellsfollowing passive uptake transfection of: Nano Lead (no Chl); GuideStrand alone (18 PS); Nano (GS: 0 PS, 19 nt); Nano (GS: 2 PS, 19 nt);Nano (GS: 4 PS, 19 nt); Nano (GS: 6 PS, 19 nt); Nano Lead (GS: 8 PS, 19nt); Nano (GS: 10 PS, 19 nt); Nano (GS: 12 PS, 19 nt); and Nano (GS: 18PS, 19 nt).

FIG. 43 is a schematic depicting guide strand chemical modificationsthat were screened for optimization.

FIG. 44 is a graph showing percent MAP4K4 expression in Hek293 cellsfollowing reverse transfection of: RISC free siRNA; GS only (2′F C andUs); GS only (2′OMe C and Us); Nano Lead (2′F C and Us); nano (GS: (3)2′OMe, positions 16-18); nano (GS: (3) 2′OMe, positions 16, 17 and 19);nano (GS: (4) 2′OMe, positions 11, 16-18); nano (GS: (10) 2′OMe, C andUs); nano (GS: (6) 2′OMe, positions 1 and 5-9); nano (GS: (3) 2′OMe,positions 1, 18 and 19); and nano (GS: (5) 2′OMe Cs).

FIG. 45 is a graph demonstrating efficacy of various chemicalmodification patterns. In particular, 2-OMe modification in positions 1and 11-18 was well tolerated. 2′OMe modifications in the seed arearesulted in a slight reduction of efficacy (but were still highlyefficient). Ribo-modifications in the seed were well tolerated. Thisdata enabled the generation of self delivering compounds with reduced orno 2′F modifications. This is significant because 2′F modifications maybe associated with toxicity in vivo.

FIG. 46 is a schematic depicting sense strand modifications.

FIG. 47 is a graph demonstrating sense strand length optimization. Asense strand length between 10-15 bases was found to be optimal in thisassay. Increasing sense strand length resulted in a reduction of passiveuptake of these compounds but may be tolerated for other compounds.Sense strands containing LNA modification demonstrated similar efficacyto non-LNA containing compounds. In some embodiments, the addition ofLNA or other thermodynamically stabilizing compounds can be beneficial,resulting in converting non-functional sequences into functionalsequences.

FIG. 48 is a graph showing percent MAP4K4 expression in HeLa cellsfollowing passive uptake transfection of: Guide Strand Alone (2′F C andU); Nano Lead; Nano Lead (No Chl); Nano (SS: 11 nt 2′OMe C and Us, Chl);Nano (SS: 11 nt, complete 2′OMe, Chl); Nano (SS: 19 nt, 2′OMe C and Us,Chl); Nano (SS: 19 nt, 2′OMe C and Us, no Chl).

FIG. 49 is a graph showing percent MAP4K4 expression in HeLa cellsfollowing passive uptake transfection of: Nano Lead (No Chl); Nano (SSno PS); Nano Lead (SS:2 PS); Nano (SS:4 PS).

FIG. 50 is a schematic depicting a sd-rxRNA'° second generation (GII)lead molecule.

FIG. 51 presents a graph indicating EC50 values for MAP4K4 silencing inthe presence of sd-rxRNA, and images depicting localization ofDY547-labeled rxRNA^(ori) and DY547-labeled sd-rxRNA.

FIG. 52 is a graph showing percent MAP4K4 expression in HeLa cells inthe presence of optimized sd-rxRNA molecules.

FIG. 53 is a graph depicting the relevance of chemistry content inoptimization of sd-rxRNA efficacy.

FIG. 54 presents schematics of sterol-type molecules and a graphrevealing that sd-rxRNA compounds are fully functional with a variety oflinker chemistries. GII asymmetric compounds were synthesized withsteroltype molecules attached through TEG and amino caproic acidlinkers. Both linkers showed identical potency. This functionalityindependent of linker chemistry indicates a significant differencebetween the molecules described herein and previously describedmolecules, and offers significant advantages for the molecules describedherein in terms of scale up and synthesis.

FIG. 55 demonstrates the stability of chemically modified sd-rxRNAcompounds in human serum in comparison to non modified RNA. Theoligonucleotides were incubated in 75% serum at 37° C. for the number ofhours indicated. The level of degradation was determined by running thesamples on non-denaturing gels and staining with SYBGR.

FIG. 56 is a graph depicting optimization of cellular uptake of sd-rxRNAthrough minimizing oligonucleotide content.

FIG. 57 is a graph showing percent MAP4K4 expression after spontaneouscellular uptake of sd-rxRNA in mouse PEC-derived macrophages, and phaseand fluorescent images showing localization of sd-rxRNA.

FIG. 58 is a graph showing percent MAP4K4 expression after spontaneouscellular uptake of sd-rxRNA (targeting) and sd-rxRNA (mismatch) in mouseprimary hepatocytes, and phase and fluorescent images showinglocalization of sd-rxRNA.

FIG. 59 presents images depicting localization of DY547-labeled sd-rxRNAdelivered to RPE cells with no formulation.

FIG. 60 is a graph showing silencing of MAP4K4 expression in RPE cellstreated with sd-rxRNA^(nano) without formulation.

FIG. 61 presents a graph and schematics of RNAi compounds showing thechemical/structural composition of highly effective sd-rxRNA compounds.Highly effective compounds were found to have the followingcharacteristics: antisense strands of 17-21 nucleotides, sense strandsof 10-15 nucleotides, single-stranded regions that contained 2-12phosphorothioate modifications, preferentially 6-8 phosphorothioatemodifications, and sense strands in which the majority of nucleotideswere 2′OMe modified, with or without phosphorothioate modification. Anylinker chemistry can be used to attach these molecules to hydrophobicmoieties such as cholesterol at the 3′ end of the sense strand. VersionGIIa-b of these RNA compounds demonstrate that elimination of 2′Fcontent has no impact on efficacy.

FIG. 62 presents a graph and schematics of RNAi compounds demonstratingthe superior performance of sd-rxRNA compounds compared to compoundspublished by Wolfrum et. al. Nature Biotech, 2007. Both generation I andII compounds (GI and GIIa) developed herein show great efficacy. Bycontrast, when the chemistry described in Wolfrum et al. (all oligoscontain cholesterol conjugated to the 3′ end of the sense strand) wasapplied to the same sequence in a context of conventional siRNA (19 bpduplex with two overhang) the compound was practically inactive. Thesedata emphasize the significance of the combination of chemicalmodifications and assymetrical molecules described herein, producinghighly effective RNA compounds.

FIG. 63 presents images showing that sd-rxRNA accumulates inside cellswhile other less effective conjugate RNAs accumulate on the surface ofcells.

FIG. 64 presents images showing that sd-rxRNA molecules, but not othermolecules, are internalized into cells within minutes.

FIG. 65 presents images demonstrating that sd-rxRNA compounds havedrastically better cellular and tissue uptake characteristics whencompared to conventional cholesterol conjugated siRNAs (such as thosepublished by Soucheck et al). FIG. 65A,B compare uptake in RPE cells,FIG. 65C,D compare uptake upon local administration to skin and FIG.65E,F compare uptake by the liver upon systemic administration. Thelevel of uptake is at least an order of magnitude higher for thesd-rxRNA compounds relative to the regular siRNA-cholesterol compounds.

FIG. 66 presents images depicting localization of rxRNA^(ori) andsd-rxRNA following local delivery.

FIG. 67 presents images depicting localization of sd-rxRNA and otherconjugate RNAs following local delivery.

FIG. 68 presents a graph revealing the results of a screen performedwith sd-rxRNAGII chemistry to identify functional compounds targetingthe SPP1 gene. Multiple effective compounds were identified, with 14131being the most effective. The compounds were added to A-549 cells andthe level of the ratio of SPP1/PPIB was determined by B-DNA after 48hours.

FIG. 69 presents a graph and several images demonstrating efficientcellular uptake of sd-rxRNA within minutes of exposure. This is a uniquecharacteristics of the sd-rxRNA compounds described herein, not observedwith any other RNAi compounds. The Soutschek et al. compound was used asa negative control.

FIG. 70 presents a graph and several images demonstrating efficientuptake and silencing of sd-rxRNA compounds in multiple cell types withmultiple sequences. In each case silencing was confirmed by looking attarget gene expression using a Branched DNA assay.

FIG. 71 presents a graph revealing that sd-rxRNA is active in thepresence and absence of serum. A slight reduction in efficacy (2-5 fold)was observed in the presence of serum. This minimal reduction inefficacy in the presence of serum differentiates the sd-rxRNA compoundsdescribed herein from previously described RNAi compounds, which had agreater reduction in efficacy, and thus creates a foundation for in vivoefficacy of the sd-rxRNA molecules described herein.

FIG. 72 presents images demonstrating efficient tissue penetration andcellular uptake upon single intradermal injection of sd-rxRNA compoundsdescribed herein. This represents a model for local delivery of sd-rxRNAcompounds as well as an effective demonstration of delivery of sd-rxRNAcompounds and silencing of genes in dermatological applications.

FIG. 73 presents images and a graph demonstrating efficient cellularuptake and in vivo silencing with sd-rxRNA following intradermalinjection.

FIG. 74 presents graphs demonstrating that sd-rxRNA compounds haveimproved blood clearance and induce effective gene silencing in vivo inthe liver upon systemic administration.

FIG. 75 presents a graph demonstrating that the presence of 5-Methyl Cin an RNAi compound resulted in an increase in potency of lipid mediatedtransfection, demonstrating that hydrophobic modification of Cs and Usin the content of RNAi compounds can be beneficial. In some embodiments,these types of modifications can be used in the context of 2′ ribosemodified bases to insure optimal stability and efficacy.

FIG. 76 presents a graph showing percent MAP4K4 expression in HeLa cellsfollowing passive uptake transfection of: Guide strand alone; Nano Lead;Nano Lead (No cholesterol); Guide Strand w/SMeC and 2′F Us Alone; NanoLead w/GS SMeC and 2′F Us; Nano Lead w/GS riboT and 5 Methyl Cs; andNano Lead w/Guide dT and 5 Methyl Cs.

FIG. 77 presents images comparing localization of sd-rxRNA and other RNAconjugates following systemic delivery to the liver.

FIG. 78 presents schematics demonstrating 5-uridyl modifications withimproved hydrophobicity characteristics. Incorporation of suchmodifications into sd-rxRNA compounds can increase cellular and tissueuptake properties. FIG. 78B presents a new type of RNAi compoundmodification which can be applied to compounds to improve cellularuptake and pharmacokinetic behavior. This type of modification, whenapplied to sd-rxRNA compounds, may contribute to making such compoundsorally available.

FIG. 79 presents schematics revealing the structures of synthesizedmodified sterol type molecules, where the length and structure of theC17 attached tail is modified. Without wishing to be bound by anytheory, the length of the C17 attached tail may contribute to improvingin vitro and in vivo efficacy of sd-rxRNA compounds.

FIG. 80 presents a schematic demonstrating the lithocholic acid route tolong side chain cholesterols.

FIG. 81 presents a schematic demonstrating a route to 5-uridylphosphoramidite synthesis.

FIG. 82 presents a schematic demonstrating synthesis of tri-functionalhydroxyprolinol linker for 3′-cholesterol attachment.

FIG. 83 presents a schematic demonstrating synthesis of solid supportfor the manufacture of a shorter asymmetric RNAi compound strand.

FIG. 84 demonstrates SPPI sd-rxRNA compound selection. Sd-rxRNAcompounds targeting SPP1 were added to A549 cells (using passivetransfection) and the level of SPP1 expression was evaluated after 48hours. Several novel compounds effective in SPP1 silencing wereidentified, the most potent of which was compound 14131.

FIG. 85 demonstrates independent validation of sd-rxRNA compounds 14116,14121, 14131, 14134, 14139, 14149, and 14152 efficacy in SPP1 silencing.

FIG. 86 demonstrates results of sd-rxRNA compound screens to identifysd-rxRNA compounds functional in CTGF knockdown.

FIG. 87 demonstrates results of sd-rxRNA compound screens to identifysd-rxRNA functional in CTGF knockdown.

FIG. 88 demonstrates a systematic screen identifying the minimal lengthof the asymmetric compounds. The passenger strand of 10-19 bases washybridized to a guide strand of 17-25 bases. In this assay, compoundswith duplex regions as short as 10 bases were found to be effective ininducing.

FIG. 89 demonstrates that positioning of the sense strand relative tothe guide strand is critical for RNAi Activity. In this assay, a bluntend was found to be optimal, a 3′ overhang was tolerated, and a 5′overhang resulted in complete loss of functionality.

FIG. 90 demonstrates that the guide strand, which has homology to thetarget only at nucleotides 2-17, resulted in effective RNAi whenhybridized with sense strands of different lengths. The compounds wereintroduced into HeLa cells via lipid mediated transfection.

FIG. 91 is a schematic depicting a panel of sterol-type molecules whichcan be used as a hydrophobic entity in place of cholesterol. In someinstances, the use of sterol-type molecules comprising longer chainsresults in generation of sd-rxRNA compounds with significantly bettercellular uptake and tissue distribution properties.

FIG. 92 presents schematics depicting a panel of hydrophobic moleculeswhich might be used as a hydrophobic entity in place of cholesterol.These list just provides representative examples; any small moleculewith substantial hydrophobicity can be used.

DETAILED DESCRIPTION

Aspects of the invention relate to methods and compositions involved ingene silencing. The invention is based at least in part on thesurprising discovery that asymmetric nucleic acid molecules with adouble stranded region of a minimal length such as 8-14 nucleotides, areeffective in silencing gene expression. Molecules with such a shortdouble stranded region have not previously been demonstrated to beeffective in mediating RNA interference. It had previously been assumedthat that there must be a double stranded region of 19 nucleotides orgreater. The molecules described herein are optimized through chemicalmodification, and in some instances through attachment of hydrophobicconjugates.

The invention is based at least in part on another surprising discoverythat asymmetric nucleic acid molecules with reduced double strandedregions are much more effectively taken up by cells compared toconventional siRNAs. These molecules are highly efficient in silencingof target gene expression and offer significant advantages overpreviously described RNAi molecules including high activity in thepresence of serum, efficient self delivery, compatibility with a widevariety of linkers, and reduced presence or complete absence of chemicalmodifications that are associated with toxicity.

In contrast to single-stranded polynucleotides, duplex polynucleotideshave been difficult to deliver to a cell as they have rigid structuresand a large number of negative charges which makes membrane transferdifficult. Unexpectedly, it was found that the polynucleotides of thepresent invention, although partially double-stranded, are recognized invivo as single-stranded and, as such, are capable of efficiently beingdelivered across cell membranes. As a result the polynucleotides of theinvention are capable in many instances of self delivery. Thus, thepolynucleotides of the invention may be formulated in a manner similarto conventional RNAi agents or they may be delivered to the cell orsubject alone (or with non-delivery type carriers) and allowed to selfdeliver. In one embodiment of the present invention, self deliveringasymmetric double-stranded RNA molecules are provided in which oneportion of the molecule resembles a conventional RNA duplex and a secondportion of the molecule is single stranded.

The polynucleotides of the invention are referred to herein as isolateddouble stranded or duplex nucleic acids, oligonucleotides orpolynucleotides, nano molecules, nano RNA, sd-rxRNA^(nano), sd-rxRNA orRNA molecules of the invention.

The oligonucleotides of the invention in some aspects have a combinationof asymmetric structures including a double stranded region and a singlestranded region of 5 nucleotides or longer, specific chemicalmodification patterns and are conjugated to lipophilic or hydrophobicmolecules. This new class of RNAi like compounds have superior efficacyin vitro and in vivo. Based on the data described herein it is believedthat the reduction in the size of the rigid duplex region in combinationwith phosphorothioate modifications applied to a single stranded regionare new and important for achieving the observed superior efficacy.Thus, the RNA molecules described herein are different in both structureand composition as well as in vitro and in vivo activity.

In a preferred embodiment the RNAi compounds of the invention comprisean asymmetric compound comprising a duplex region (required forefficient RISC entry of 10-15 bases long) and single stranded region of4-12 nucleotides long; with a 13 nucleotide duplex. A 6 nucleotidesingle stranded region is preferred in some embodiments. The singlestranded region of the new RNAi compounds also comprises 2-12phosphorothioate internucleotide linkages (referred to asphosphorothioate modifications). 6-8 phosphorothioate internucleotidelinkages are preferred in some embodiments. Additionally, the RNAicompounds of the invention also include a unique chemical modificationpattern, which provides stability and is compatible with RISC entry. Thecombination of these elements has resulted in unexpected propertieswhich are highly useful for delivery of RNAi reagents in vitro and invivo.

The chemically modification pattern, which provides stability and iscompatible with RISC entry includes modifications to the sense, orpassenger, strand as well as the antisense, or guide, strand. Forinstance the passenger strand can be modified with any chemical entitieswhich confirm stability and do not interfere with activity. Suchmodifications include 2′ ribo modifications (O-methyl, 2′ F, 2 deoxy andothers) and backbone modification like phosphorothioate modifications. Apreferred chemical modification pattern in the passenger strand includesOmethyl modification of C and U nucleotides within the passenger strandor alternatively the passenger strand may be completely Omethylmodified.

The guide strand, for example, may also be modified by any chemicalmodification which confirms stability without interfering with RISCentry. A preferred chemical modification pattern in the guide strandincludes the majority of C and U nucleotides being 2′ F modified and the5′ end being phosphorylated. Another preferred chemical modificationpattern in the guide strand includes 2′ Omethyl modification of position1 and C/U in positions 11-18 and 5′ end chemical phosphorylation. Yetanother preferred chemical modification pattern in the guide strandincludes 2′ Omethyl modification of position 1 and C/U in positions11-18 and 5′ end chemical phosphorylation and 2′F modification of C/U inpositions 2-10.

It was surprisingly discovered according to the invention that theabove-described chemical modification patterns of the oligonucleotidesof the invention are well tolerated and actually improved efficacy ofasymmetric RNAi compounds. See, for instance, FIG. 22.

It was also demonstrated experimentally herein that the combination ofmodifications to RNAi when used together in a polynucleotide results inthe achievement of optimal efficacy in passive uptake of the RNAi.Elimination of any of the described components (Guide strandstabilization, phosphorothioate stretch, sense strand stabilization andhydrophobic conjugate) or increase in size results in sub-optimalefficacy and in some instances complete lost of efficacy. Thecombination of elements results in development of compound, which isfully active following passive delivery to cells such as HeLa cells.(FIG. 23). The degree to which the combination of elements results inefficient self delivery of RNAi molecules was completely unexpected.

The data shown in FIGS. 26, 27 and 43 demonstrated the importance of thevarious modifications to the RNAi in achieving stabilization andactivity. For instance, FIG. 26 demonstrates that use off asymmetricconfiguration is important in getting efficacy in passive uptake. Whenthe same chemical composition is applied to compounds of traditionalconfigurations (19-21 bases duplex and 25 mer duplex) the efficacy wasdrastically decreased in a length dependent manner. FIG. 27 demonstrateda systematic screen of the impact of phosphorothioate chemicalmodifications on activity. The sequence, structure, stabilizationchemical modifications, hydrophobic conjugate were kept constant andcompound phosphorothioate content was varied (from 0 to 18 PS bond).Both compounds having no phosphorothioate linkages and having 18phosphorothioate linkages were completely inactive in passive uptake.Compounds having 2-16 phosphorothioate linkages were active, withcompounds having 4-10 phosphorothioate being the most active compounds.

The data in the Examples presented below demonstrates high efficacy ofthe oligonucleotides of the invention both in vitro in variety of celltypes (supporting data) and in vivo upon local and systemicadministration. For instance, the data compares the ability of severalcompetitive RNAi molecules having different chemistries to silence agene. Comparison of sd-rxRNA (oligonucleotides of the invention) withRNAs described in Soucheck et al. and Wolfrum at al., as applied to thesame targeting region, demonstrated that only sd-rxRNA chemistry showeda significant functionality in passive uptake. The composition of theinvention achieved EC50 values of 10-50 μM. This level of efficacy isun-attainable with conventional chemistries like those described inSauthceck at al and Accell. Similar comparisons were made in othersystems, such as in vitro (RPE cell line), in vivo upon localadministration (wounded skin) and systemic (50 mg/kg) as well as othergenes (FIGS. 65 and 68). In each case the oligonucleotides of theinvention achieved better results. FIG. 64 includes data demonstratingefficient cellular uptake and resulting silencing by sd-rxRNA compoundsonly after 1 minute of exposure. Such an efficacy is unique to thiscomposition and have not been seen with other types of molecules in thisclass. FIG. 70 demonstrates efficient uptake and silencing of sd-rxRNAcompounds in multiple cell types with multiple sequences. The sd-rxRNAcompounds are also active in cells in presence and absence of serum andother biological liquids. FIG. 71 demonstrates only a slight reductionin activity in the presence of serum. This ability to function inbiologically aggressive environment effectively further differentiatessd-rxRNA compounds from other compounds described previously in thisgroup, like Accell and Soucheck et al, in which uptake is drasticallyinhibited in a presence of serum.

Significant amounts of data also demonstrate the in vivo efficacy of thecompounds of the invention. For instance FIGS. 72-74 involve multipleroutes of in vivo delivery of the compounds of the invention resultingin significant activity. FIG. 72, for example, demonstrates efficienttissue penetration and cellular uptake upon single intradermalinjection. This is a model for local delivery of sd-rxRNA compounds aswell as an effective delivery mode for sd-rxRNA compounds and silencinggenes in any dermatology applications. FIG. 73 demonstrated efficienttissue penetration, cellular uptake and silencing upon local in vivointradermal injection of sd-rxRNA compounds. The data of FIG. 74demonstrate that sd-rxRNA compounds result in highly effective liveruptake upon IV administration. Comparison to Souicheck at al moleculeshowed that the level of liver uptake at identical dose level was quitesurprisingly, at least 50 fold higher with the sd-rxRNA compound thanthe Souicheck at al molecule.

The sd-rxRNA can be further improved in some instances by improving thehydrophobicity of compounds using of novel types of chemistries. Forexample one chemistry is related to use of hydrophobic basemodifications. Any base in any position might be modified, as long asmodification results in an increase of the partition coefficient of thebase. The preferred locations for modification chemistries are positions4 and 5 of the pyrimidines. The major advantage of these positions is(a) ease of synthesis and (b) lack of interference with base-pairing andA form helix formation, which are essential for RISC complex loading andtarget recognition. Examples of these chemistries is shown in FIGS.75-83. A version of sd-rxRNA compounds where multiple deoxy Uridines arepresent without interfering with overall compound efficacy was used. Inaddition major improvement in tissue distribution and cellular uptakemight be obtained by optimizing the structure of the hydrophobicconjugate. In some of the preferred embodiment the structure of sterolis modified to alter (increase/decrease) C17 attached chain. This typeof modification results in significant increase in cellular uptake andimprovement of tissue uptake prosperities in vivo.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

Thus, aspects of the invention relate to isolated double strandednucleic acid molecules comprising a guide (antisense) strand and apassenger (sense) strand. As used herein, the term “double-stranded”refers to one or more nucleic acid molecules in which at least a portionof the nucleomonomers are complementary and hydrogen bond to form adouble-stranded region. In some embodiments, the length of the guidestrand ranges from 16-29 nucleotides long. In certain embodiments, theguide strand is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or29 nucleotides long. The guide strand has complementarity to a targetgene. Complementarity between the guide strand and the target gene mayexist over any portion of the guide strand. Complementarity as usedherein may be perfect complementarity or less than perfectcomplementarity as long as the guide strand is sufficientlycomplementary to the target that it mediates RNAi. In some embodimentscomplementarity refers to less than 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%,or 1% mismatch between the guide strand and the target. Perfectcomplementarity refers to 100% complementarity. Thus the invention hasthe advantage of being able to tolerate sequence variations that mightbe expected due to genetic mutation, strain polymorphism, orevolutionary divergence. For example, siRNA sequences with insertions,deletions, and single point mutations relative to the target sequencehave also been found to be effective for inhibition. Moreover, not allpositions of a siRNA contribute equally to target recognition.Mismatches in the center of the siRNA are most critical and essentiallyabolish target RNA cleavage. Mismatches upstream of the center orupstream of the cleavage site referencing the antisense strand aretolerated but significantly reduce target RNA cleavage. Mismatchesdownstream of the center or cleavage site referencing the antisensestrand, preferably located near the 3′ end of the antisense strand, e.g.1, 2, 3, 4, 5 or 6 nucleotides from the 3′ end of the antisense strand,are tolerated and reduce target RNA cleavage only slightly.

While not wishing to be bound by any particular theory, in someembodiments, the guide strand is at least 16 nucleotides in length andanchors the Argonaute protein in RISC. In some embodiments, when theguide strand loads into RISC it has a defined seed region and targetmRNA cleavage takes place across from position 10-11 of the guidestrand. In some embodiments, the 5′ end of the guide strand is or isable to be phosphorylated. The nucleic acid molecules described hereinmay be referred to as minimum trigger RNA.

In some embodiments, the length of the passenger strand ranges from 8-14nucleotides long. In certain embodiments, the passenger strand is 8, 9,10, 11, 12, 13 or 14 nucleotides long. The passenger strand hascomplementarity to the guide strand. Complementarity between thepassenger strand and the guide strand can exist over any portion of thepassenger or guide strand. In some embodiments, there is 100%complementarity between the guide and passenger strands within thedouble stranded region of the molecule.

Aspects of the invention relate to double stranded nucleic acidmolecules with minimal double stranded regions. In some embodiments theregion of the molecule that is double stranded ranges from 8-14nucleotides long. In certain embodiments, the region of the moleculethat is double stranded is 8, 9, 10, 11, 12, 13 or 14 nucleotides long.In certain embodiments the double stranded region is 13 nucleotideslong. There can be 100% complementarity between the guide and passengerstrands, or there may be one or more mismatches between the guide andpassenger strands. In some embodiments, on one end of the doublestranded molecule, the molecule is either blunt-ended or has aone-nucleotide overhang. The single stranded region of the molecule isin some embodiments between 4-12 nucleotides long. For example thesingle stranded region can be 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotideslong. However, in certain embodiments, the single stranded region canalso be less than 4 or greater than 12 nucleotides long. In certainembodiments, the single stranded region is 6 nucleotides long.

RNAi constructs associated with the invention can have a thermodynamicstability (AG) of less than −13 kkal/mol. In some embodiments, thethermodynamic stability (AG) is less than −20 kkal/mol. In someembodiments there is a loss of efficacy when (AG) goes below −21kkal/mol. In some embodiments a (AG) value higher than −13 kkal/mol iscompatible with aspects of the invention. Without wishing to be bound byany theory, in some embodiments a molecule with a relatively higher (AG)value may become active at a relatively higher concentration, while amolecule with a relatively lower (AG) value may become active at arelatively lower concentration. In some embodiments, the (AG) value maybe higher than −9 kkcal/mol. The gene silencing effects mediated by theRNAi constructs associated with the invention, containing minimal doublestranded regions, are unexpected because molecules of almost identicaldesign but lower thermodynamic stability have been demonstrated to beinactive (Rana et al 2004).

Without wishing to be bound by any theory, results described hereinsuggest that a stretch of 8-10 bp of dsRNA or dsDNA will be structurallyrecognized by protein components of RISC or co-factors of RISC.Additionally, there is a free energy requirement for the triggeringcompound that it may be either sensed by the protein components and/orstable enough to interact with such components so that it may be loadedinto the Argonaute protein. If optimal thermodynamics are present andthere is a double stranded portion that is preferably at least 8nucleotides then the duplex will be recognized and loaded into the RNAimachinery.

In some embodiments, thermodynamic stability is increased through theuse of LNA bases. In some embodiments, additional chemical modificationsare introduced. Several non-limiting examples of chemical modificationsinclude: 5′ Phosphate, 2′-O-methyl, 2′-O-ethyl, 2′-fluoro,ribothymidine, C-5 propynyl-dC (pdC) and C-5 propynyl-dU (pdU); C-5propynyl-C (pC) and C-5 propynyl-U (pU); 5-methyl C, 5-methyl U,5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine),5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine and MGB (minor groovebinder). It should be appreciated that more than one chemicalmodification can be combined within the same molecule.

Molecules associated with the invention are optimized for increasedpotency and/or reduced toxicity. For example, nucleotide length of theguide and/or passenger strand, and/or the number of phosphorothioatemodifications in the guide and/or passenger strand, can in some aspectsinfluence potency of the RNA molecule, while replacing 2′-fluoro (2′F)modifications with 2′-O-methyl (2′OMe) modifications can in some aspectsinfluence toxicity of the molecule. Specifically, reduction in 2′Fcontent of a molecule is predicted to reduce toxicity of the molecule.The Examples section presents molecules in which 2′F modifications havebeen eliminated, offering an advantage over previously described RNAicompounds due to a predicted reduction in toxicity. Furthermore, thenumber of phosphorothioate modifications in an RNA molecule caninfluence the uptake of the molecule into a cell, for example theefficiency of passive uptake of the molecule into a cell. Preferredembodiments of molecules described herein have no 2′F modification andyet are characterized by equal efficacy in cellular uptake and tissuepenetration. Such molecules represent a significant improvement overprior art, such as molecules described by Accell and Wolfrum, which areheavily modified with extensive use of 2′F.

In some embodiments, a guide strand is approximately 18-19 nucleotidesin length and has approximately 2-14 phosphate modifications. Forexample, a guide strand can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or more than 14 nucleotides that are phosphate-modified. Theguide strand may contain one or more modifications that confer increasedstability without interfering with RISC entry. The phosphate modifiednucleotides, such as phosphorothioate modified nucleotides, can be atthe 3′ end, 5′ end or spread throughout the guide strand. In someembodiments, the 3′ terminal 10 nucleotides of the guide strand contains1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioate modified nucleotides.The guide strand can also contain 2′F and/or 2′OMe modifications, whichcan be located throughout the molecule. In some embodiments, thenucleotide in position one of the guide strand (the nucleotide in themost 5′ position of the guide strand) is 2′OMe modified and/orphosphorylated. C and U nucleotides within the guide strand can be 2′Fmodified. For example, C and U nucleotides in positions 2-10 of a 19 ntguide strand (or corresponding positions in a guide strand of adifferent length) can be 2′F modified. C and U nucleotides within theguide strand can also be 2′OMe modified. For example, C and Unucleotides in positions 11-18 of a 19 nt guide strand (or correspondingpositions in a guide strand of a different length) can be 2′OMemodified. In some embodiments, the nucleotide at the most 3′ end of theguide strand is unmodified. In certain embodiments, the majority of Csand Us within the guide strand are 2′F modified and the 5′ end of theguide strand is phosphorylated. In other embodiments, position 1 and theCs or Us in positions 11-18 are 2′OMe modified and the 5′ end of theguide strand is phosphorylated. In other embodiments, position 1 and theCs or Us in positions 11-18 are 2′OMe modified, the 5′ end of the guidestrand is phosphorylated, and the Cs or Us in position 2-10 are 2′Fmodified.

In some aspects, an optimal passenger strand is approximately 11-14nucleotides in length. The passenger strand may contain modificationsthat confer increased stability. One or more nucleotides in thepassenger strand can be 2′OMe modified. In some embodiments, one or moreof the C and/or U nucleotides in the passenger strand is 2′OMe modified,or all of the C and U nucleotides in the passenger strand are 2′OMemodified. In certain embodiments, all of the nucleotides in thepassenger strand are 2′OMe modified. One or more of the nucleotides onthe passenger strand can also be phosphate-modified such asphosphorothioate modified. The passenger strand can also contain 2′ribo, 2′F and 2 deoxy modifications or any combination of the above. Asdemonstrated in the Examples, chemical modification patterns on both theguide and passenger strand are well tolerated and a combination ofchemical modifications is shown herein to lead to increased efficacy andself-delivery of RNA molecules.

Aspects of the invention relate to RNAi constructs that have extendedsingle-stranded regions relative to double stranded regions, as comparedto molecules that have been used previously for RNAi. The singlestranded region of the molecules may be modified to promote cellularuptake or gene silencing. In some embodiments, phosphorothioatemodification of the single stranded region influences cellular uptakeand/or gene silencing. The region of the guide strand that isphosphorothioate modified can include nucleotides within both the singlestranded and double stranded regions of the molecule. In someembodiments, the single stranded region includes 2-12 phosphorothioatemodifications. For example, the single stranded region can include 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphorothioate modifications. In someinstances, the single stranded region contains 6-8 phosphorothioatemodifications.

Molecules associated with the invention are also optimized for cellularuptake. In RNA molecules described herein, the guide and/or passengerstrands can be attached to a conjugate. In certain embodiments theconjugate is hydrophobic. The hydrophobic conjugate can be a smallmolecule with a partition coefficient that is higher than 10. Theconjugate can be a sterol-type molecule such as cholesterol, or amolecule with an increased length polycarbon chain attached to C17, andthe presence of a conjugate can influence the ability of an RNA moleculeto be taken into a cell with or without a lipid transfection reagent.The conjugate can be attached to the passenger or guide strand through ahydrophobic linker. In some embodiments, a hydrophobic linker is 5-12Cin length, and/or is hydroxypyrrolidine-based. In some embodiments, ahydrophobic conjugate is attached to the passenger strand and the CUresidues of either the passenger and/or guide strand are modified. Insome embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%or 95% of the CU residues on the passenger strand and/or the guidestrand are modified. In some aspects, molecules associated with theinvention are self-delivering (sd). As used herein, “self-delivery”refers to the ability of a molecule to be delivered into a cell withoutthe need for an additional delivery vehicle such as a transfectionreagent.

Aspects of the invention relate to selecting molecules for use in RNAi.Based on the data described herein, molecules that have a doublestranded region of 8-14 nucleotides can be selected for use in RNAi. Insome embodiments, molecules are selected based on their thermodynamicstability (AG). In some embodiments, molecules will be selected thathave a (AG) of less than −13 kkal/mol. For example, the (AG) value maybe −13, −14, −15, −16, −17, −18, −19, −21, −22 or less than −22kkal/mol. In other embodiments, the (AG) value may be higher than −13kkal/mol. For example, the (AG) value may be −12, −11, −10, −9, −8, −7or more than −7 kkal/mol. It should be appreciated that ΔG can becalculated using any method known in the art. In some embodiments ΔG iscalculated using Mfold, available through the Mfold internet site(http://mvold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi). Methods forcalculating ΔG are described in, and are incorporated by reference from,the following references: Zuker, M. (2003) Nucleic Acids Res.,31(13):3406-15; Mathews, D. H., Sabina, J., Zuker, M. and Turner, D. H.(1999) J. Mol. Biol. 288:911-940; Mathews, D. H., Disney, M. D., Childs,J. L., Schroeder, S. J., Zuker, M., and Turner, D. H. (2004) Proc. Natl.Acad. Sci. 101:7287-7292; Duan, S., Mathews, D. H., and Turner, D. H.(2006) Biochemistry 45:9819-9832; Wuchty, S., Fontana, W., Hofacker, I.L., and Schuster, P. (1999) Biopolymers 49:145-165.

Aspects of the invention relate to using nucleic acid moleculesdescribed herein, with minimal double stranded regions and/or with a(ΔG) of less than −13 kkal/mol, for gene silencing. RNAi molecules canbe administered in vivo or in vitro, and gene silencing effects can beachieved in vivo or in vitro.

In certain embodiments, the polynucleotide contains 5′- and/or 3′-endoverhangs. The number and/or sequence of nucleotides overhang on one endof the polynucleotide may be the same or different from the other end ofthe polynucleotide. In certain embodiments, one or more of the overhangnucleotides may contain chemical modification(s), such asphosphorothioate or 2′-OMe modification.

In certain embodiments, the polynucleotide is unmodified. In otherembodiments, at least one nucleotide is modified. In furtherembodiments, the modification includes a 2′-H or 2′-modified ribosesugar at the 2nd nucleotide from the 5′-end of the guide sequence. The“2nd nucleotide” is defined as the second nucleotide from the 5′-end ofthe polynucleotide.

As used herein, “2′-modified ribose sugar” includes those ribose sugarsthat do not have a 2′-OH group. “2′-modified ribose sugar” does notinclude 2′-deoxyribose (found in unmodified canonical DNA nucleotides).For example, the 2′-modified ribose sugar may be 2′-O-alkyl nucleotides,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy nucleotides, or combinationthereof.

In certain embodiments, the 2′-modified nucleotides are pyrimidinenucleotides (e.g., C/U). Examples of 2′-O-alkyl nucleotides include2′-O-methyl nucleotides, or 2′-O-allyl nucleotides.

In certain embodiments, the miniRNA polynucleotide of the invention withthe above-referenced 5′-end modification exhibits significantly (e.g.,at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90% or more) less “off-target” gene silencing when compared tosimilar constructs without the specified 5′-end modification, thusgreatly improving the overall specificity of the RNAi reagent ortherapeutics.

As used herein, “off-target” gene silencing refers to unintended genesilencing due to, for example, spurious sequence homology between theantisense (guide) sequence and the unintended target mRNA sequence.

According to this aspect of the invention, certain guide strandmodifications further increase nuclease stability, and/or lowerinterferon induction, without significantly decreasing RNAi activity (orno decrease in RNAi activity at all).

In some embodiments, the 5′-stem sequence may comprise a 2′-modifiedribose sugar, such as 2′-O-methyl modified nucleotide, at the 2^(nd)nucleotide on the 5′-end of the polynucleotide and, in some embodiments,no other modified nucleotides. The hairpin structure having suchmodification may have enhanced target specificity or reduced off-targetsilencing compared to a similar construct without the 2′-O-methylmodification at said position.

Certain combinations of specific 5′-stem sequence and 3′-stem sequencemodifications may result in further unexpected advantages, as partlymanifested by enhanced ability to inhibit target gene expression,enhanced serum stability, and/or increased target specificity, etc.

In certain embodiments, the guide strand comprises a 2′-O-methylmodified nucleotide at the 2^(nd) nucleotide on the 5′-end of the guidestrand and no other modified nucleotides.

In other aspects, the miniRNA structures of the present inventionmediates sequence-dependent gene silencing by a microRNA mechanism. Asused herein, the term “microRNA” (“miRNA”), also referred to in the artas “small temporal RNAs” (“stRNAs”), refers to a small (10-50nucleotide) RNA which are genetically encoded (e.g., by viral,mammalian, or plant genomes) and are capable of directing or mediatingRNA silencing. An “miRNA disorder” shall refer to a disease or disordercharacterized by an aberrant expression or activity of an miRNA.

microRNAs are involved in down-regulating target genes in criticalpathways, such as development and cancer, in mice, worms and mammals.Gene silencing through a microRNA mechanism is achieved by specific yetimperfect base-pairing of the miRNA and its target messenger RNA (mRNA).Various mechanisms may be used in microRNA-mediated down-regulation oftarget mRNA expression.

miRNAs are noncoding RNAs of approximately 22 nucleotides which canregulate gene expression at the post transcriptional or translationallevel during plant and animal development. One common feature of miRNAsis that they are all excised from an approximately 70 nucleotideprecursor RNA stem-loop termed pre-miRNA, probably by Dicer, an RNaseIII-type enzyme, or a homolog thereof. Naturally-occurring miRNAs areexpressed by endogenous genes in vivo and are processed from a hairpinor stem-loop precursor (pre-miRNA or pri-miRNAs) by Dicer or otherRNAses. miRNAs can exist transiently in vivo as a double-stranded duplexbut only one strand is taken up by the RISC complex to direct genesilencing.

In some embodiments a version of sd-rxRNA compounds, which are effectivein cellular uptake and inhibiting of miRNA activity are described.Essentially the compounds are similar to RISC entering version but largestrand chemical modification patterns are optimized in the way to blockcleavage and act as an effective inhibitor of the RISC action. Forexample, the compound might be completely or mostly Omethyl modifiedwith the PS content described previously. For these types of compoundsthe 5′ phosphorilation is not necessary. The presence of double strandedregion is preferred as it is promotes cellular uptake and efficient RISCloading.

Another pathway that uses small RNAs as sequence-specific regulators isthe RNA interference (RNAi) pathway, which is an evolutionarilyconserved response to the presence of double-stranded RNA (dsRNA) in thecell. The dsRNAs are cleaved into ˜20-base pair (bp) duplexes ofsmall-interfering RNAs (siRNAs) by Dicer. These small RNAs get assembledinto multiprotein effector complexes called RNA-induced silencingcomplexes (RISCs). The siRNAs then guide the cleavage of target mRNAswith perfect complementarity.

Some aspects of biogenesis, protein complexes, and function are sharedbetween the siRNA pathway and the miRNA pathway. The subjectsingle-stranded polynucleotides may mimic the dsRNA in the siRNAmechanism, or the microRNA in the miRNA mechanism.

In certain embodiments, the modified RNAi constructs may have improvedstability in serum and/or cerebral spinal fluid compared to anunmodified RNAi constructs having the same sequence.

In certain embodiments, the structure of the RNAi construct does notinduce interferon response in primary cells, such as mammalian primarycells, including primary cells from human, mouse and other rodents, andother non-human mammals. In certain embodiments, the RNAi construct mayalso be used to inhibit expression of a target gene in an invertebrateorganism.

To further increase the stability of the subject constructs in vivo, the3′-end of the hairpin structure may be blocked by protective group(s).For example, protective groups such as inverted nucleotides, invertedabasic moieties, or amino-end modified nucleotides may be used. Invertednucleotides may comprise an inverted deoxynucleotide. Inverted abasicmoieties may comprise an inverted deoxyabasic moiety, such as a3′,3′-linked or 5′,5′-linked deoxyabasic moiety.

The RNAi constructs of the invention are capable of inhibiting thesynthesis of any target protein encoded by target gene(s). The inventionincludes methods to inhibit expression of a target gene either in a cellin vitro, or in vivo. As such, the RNAi constructs of the invention areuseful for treating a patient with a disease characterized by theoverexpression of a target gene.

The target gene can be endogenous or exogenous (e.g., introduced into acell by a virus or using recombinant DNA technology) to a cell. Suchmethods may include introduction of RNA into a cell in an amountsufficient to inhibit expression of the target gene. By way of example,such an RNA molecule may have a guide strand that is complementary tothe nucleotide sequence of the target gene, such that the compositioninhibits expression of the target gene.

The invention also relates to vectors expressing the nucleic acids ofthe invention, and cells comprising such vectors or the nucleic acids.The cell may be a mammalian cell in vivo or in culture, such as a humancell.

The invention further relates to compositions comprising the subjectRNAi constructs, and a pharmaceutically acceptable carrier or diluent.

Another aspect of the invention provides a method for inhibiting theexpression of a target gene in a mammalian cell, comprising contactingthe mammalian cell with any of the subject RNAi constructs.

The method may be carried out in vitro, ex vivo, or in vivo, in, forexample, mammalian cells in culture, such as a human cell in culture.

The target cells (e.g., mammalian cell) may be contacted in the presenceof a delivery reagent, such as a lipid (e.g., a cationic lipid) or aliposome.

Another aspect of the invention provides a method for inhibiting theexpression of a target gene in a mammalian cell, comprising contactingthe mammalian cell with a vector expressing the subject RNAi constructs.

In one aspect of the invention, a longer duplex polynucleotide isprovided, including a first polynucleotide that ranges in size fromabout 16 to about 30 nucleotides; a second polynucleotide that ranges insize from about 26 to about 46 nucleotides, wherein the firstpolynucleotide (the antisense strand) is complementary to both thesecond polynucleotide (the sense strand) and a target gene, and whereinboth polynucleotides form a duplex and wherein the first polynucleotidecontains a single stranded region longer than 6 bases in length and ismodified with alternative chemical modification pattern, and/or includesa conjugate moiety that facilitates cellular delivery. In thisembodiment, between about 40% to about 90% of the nucleotides of thepassenger strand between about 40% to about 90% of the nucleotides ofthe guide strand, and between about 40% to about 90% of the nucleotidesof the single stranded region of the first polynucleotide are chemicallymodified nucleotides.

In an embodiment, the chemically modified nucleotide in thepolynucleotide duplex may be any chemically modified nucleotide known inthe art, such as those discussed in detail above. In a particularembodiment, the chemically modified nucleotide is selected from thegroup consisting of 2′ F modified nucleotides,2′-β-methyl modified and2′ deoxy nucleotides. In another particular embodiment, the chemicallymodified nucleotides results from “hydrophobic modifications” of thenucleotide base. In another particular embodiment, the chemicallymodified nucleotides are phosphorothioates. In an additional particularembodiment, chemically modified nucleotides are combination ofphosphorothioates, 2′-O-methyl, 2′ deoxy, hydrophobic modifications andphosphorothioates. As these groups of modifications refer tomodification of the ribose ring, back bone and nucleotide, it isfeasible that some modified nucleotides will carry a combination of allthree modification types.

In another embodiment, the chemical modification is not the same acrossthe various regions of the duplex. In a particular embodiment, the firstpolynucleotide (the passenger strand), has a large number of diversechemical modifications in various positions. For this polynucleotide upto 90% of nucleotides might be chemically modified and/or havemismatches introduced.

In another embodiment, chemical modifications of the first or secondpolynucleotide include, but not limited to, 5′ position modification ofUridine and Cytosine (4-pyridyl, 2-pyridyl, indolyl, phenyl (C₆H₅OH);tryptophanyl (C8H6N)CH2CH(NH2)CO), isobutyl, butyl, aminobenzyl; phenyl;naphthyl, etc), where the chemical modification might alter base pairingcapabilities of a nucleotide. For the guide strand an important featureof this aspect of the invention is the position of the chemicalmodification relative to the 5′ end of the antisense and sequence. Forexample, chemical phosphorylation of the 5′ end of the guide strand isusually beneficial for efficacy. O-methyl modifications in the seedregion of the sense strand (position 2-7 relative to the 5′ end) are notgenerally well tolerated, whereas 2′F and deoxy are well tolerated. Themid part of the guide strand and the 3′ end of the guide strand are morepermissive in a type of chemical modifications applied. Deoxymodifications are not tolerated at the 3′ end of the guide strand.

A unique feature of this aspect of the invention involves the use ofhydrophobic modification on the bases. In one embodiment, thehydrophobic modifications are preferably positioned near the 5′ end ofthe guide strand, in other embodiments, they localized in the middle ofthe guides strand, in other embodiment they localized at the 3′ end ofthe guide strand and yet in another embodiment they are distributedthought the whole length of the polynucleotide. The same type ofpatterns is applicable to the passenger strand of the duplex.

The other part of the molecule is a single stranded region. The singlestranded region is expected to range from 7 to 40 nucleotides.

In one embodiment, the single stranded region of the firstpolynucleotide contains modifications selected from the group consistingof between 40% and 90% hydrophobic base modifications, between 40%-90%phosphorothioates, between 40%-90% modification of the ribose moiety,and any combination of the preceding.

Efficiency of guide strand (first polynucleotide) loading into the RISCcomplex might be altered for heavily modified polynucleotides, so in oneembodiment, the duplex polynucleotide includes a mismatch betweennucleotide 9, 11, 12, 13, or 14 on the guide strand (firstpolynucleotide) and the opposite nucleotide on the sense strand (secondpolynucleotide) to promote efficient guide strand loading.

More detailed aspects of the invention are described in the sectionsbelow.

Duplex Characteristics

Double-stranded oligonucleotides of the invention may be formed by twoseparate complementary nucleic acid strands. Duplex formation can occureither inside or outside the cell containing the target gene.

As used herein, the term “duplex” includes the region of thedouble-stranded nucleic acid molecule(s) that is (are) hydrogen bondedto a complementary sequence. Double-stranded oligonucleotides of theinvention may comprise a nucleotide sequence that is sense to a targetgene and a complementary sequence that is antisense to the target gene.The sense and antisense nucleotide sequences correspond to the targetgene sequence, e.g., are identical or are sufficiently identical toeffect target gene inhibition (e.g., are about at least about 98%identical, 96% identical, 94%, 90% identical, 85% identical, or 80%identical) to the target gene sequence.

In certain embodiments, the double-stranded oligonucleotide of theinvention is double-stranded over its entire length, i.e., with nooverhanging single-stranded sequence at either end of the molecule,i.e., is blunt-ended. In other embodiments, the individual nucleic acidmolecules can be of different lengths. In other words, a double-strandedoligonucleotide of the invention is not double-stranded over its entirelength. For instance, when two separate nucleic acid molecules are used,one of the molecules, e.g., the first molecule comprising an antisensesequence, can be longer than the second molecule hybridizing thereto(leaving a portion of the molecule single-stranded). Likewise, when asingle nucleic acid molecule is used a portion of the molecule at eitherend can remain single-stranded.

In one embodiment, a double-stranded oligonucleotide of the inventioncontains mismatches and/or loops or bulges, but is double-stranded overat least about 70% of the length of the oligonucleotide. In anotherembodiment, a double-stranded oligonucleotide of the invention isdouble-stranded over at least about 80% of the length of theoligonucleotide. In another embodiment, a double-strandedoligonucleotide of the invention is double-stranded over at least about90%-95% of the length of the oligonucleotide. In another embodiment, adouble-stranded oligonucleotide of the invention is double-stranded overat least about 96%-98% of the length of the oligonucleotide. In certainembodiments, the double-stranded oligonucleotide of the inventioncontains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 mismatches.

Modifications

The nucleotides of the invention may be modified at various locations,including the sugar moiety, the phosphodiester linkage, and/or the base.

Sugar moieties include natural, unmodified sugars, e.g., monosaccharide(such as pentose, e.g., ribose, deoxyribose), modified sugars and sugaranalogs. In general, possible modifications of nucleomonomers,particularly of a sugar moiety, include, for example, replacement of oneor more of the hydroxyl groups with a halogen, a heteroatom, analiphatic group, or the functionalization of the hydroxyl group as anether, an amine, a thiol, or the like.

One particularly useful group of modified nucleomonomers are 2′-O-methylnucleotides. Such 2′-O-methyl nucleotides may be referred to as“methylated,” and the corresponding nucleotides may be made fromunmethylated nucleotides followed by alkylation or directly frommethylated nucleotide reagents. Modified nucleomonomers may be used incombination with unmodified nucleomonomers. For example, anoligonucleotide of the invention may contain both methylated andunmethylated nucleomonomers.

Some exemplary modified nucleomonomers include sugar- orbackbone-modified ribonucleotides. Modified ribonucleotides may containa non-naturally occurring base (instead of a naturally occurring base),such as uridines or cytidines modified at the 5′-position, e.g.,5′-(2-amino)propyl uridine and 5′-bromo uridine; adenosines andguanosines modified at the 8-position, e.g., 8-bromo guanosine; deazanucleotides, e.g., 7-deaza-adenosine; and N-alkylated nucleotides, e.g.,N6-methyl adenosine. Also, sugar-modified ribonucleotides may have the2′-OH group replaced by a H, alxoxy (or OR), R or alkyl, halogen, SH,SR, amino (such as NH₂, NHR, NR₂), or CN group, wherein R is loweralkyl, alkenyl, or alkynyl.

Modified ribonucleotides may also have the phosphodiester groupconnecting to adjacent ribonucleotides replaced by a modified group,e.g., of phosphorothioate group. More generally, the various nucleotidemodifications may be combined. Although the antisense (guide) strand maybe substantially identical to at least a portion of the target gene (orgenes), at least with respect to the base pairing properties, thesequence need not be perfectly identical to be useful, e.g., to inhibitexpression of a target gene's phenotype. Generally, higher homology canbe used to compensate for the use of a shorter antisense gene. In somecases, the antisense strand generally will be substantially identical(although in antisense orientation) to the target gene.

The use of 2′-O-methyl modified RNA may also be beneficial incircumstances in which it is desirable to minimize cellular stressresponses. RNA having 2′-O-methyl nucleomonomers may not be recognizedby cellular machinery that is thought to recognize unmodified RNA. Theuse of 2′-O-methylated or partially 2′-O-methylated RNA may avoid theinterferon response to double-stranded nucleic acids, while maintainingtarget RNA inhibition. This may be useful, for example, for avoiding theinterferon or other cellular stress responses, both in short RNAi (e.g.,siRNA) sequences that induce the interferon response, and in longer RNAisequences that may induce the interferon response.

Overall, modified sugars may include D-ribose, 2′-O-alkyl (including2′-β-methyl and 2′-O-ethyl), i.e., 2′-alkoxy, 2′-amino, 2′-S-alkyl,2′-halo (including 2′-fluoro), 2′-methoxyethoxy, 2′-allyloxy(—OCH₂CH═CH₂), 2′-propargyl, 2′-propyl, ethynyl, ethenyl, propenyl, andcyano and the like. In one embodiment, the sugar moiety can be a hexoseand incorporated into an oligonucleotide as described (Augustyns, K., etal., Nucl. Acids. Res. 18:4711 (1992)). Exemplary nucleomonomers can befound, e.g., in U.S. Pat. No. 5,849,902, incorporated by referenceherein.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.In certain embodiments, a straight chain or branched chain alkyl has 6or fewer carbon atoms in its backbone (e.g., C₁-C₆ for straight chain,C₃-C₆ for branched chain), and more preferably 4 or fewer. Likewise,preferred cycloalkyls have from 3-8 carbon atoms in their ringstructure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₁-C₆ includes alkyl groups containing 1 to 6 carbonatoms.

Moreover, unless otherwise specified, the term alkyl includes both“unsubstituted alkyls” and “substituted alkyls,” the latter of whichrefers to alkyl moieties having independently selected substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above. An “alkylaryl” or an “arylalkyl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl”also includes the side chains of natural and unnatural amino acids. Theterm “n-alkyl” means a straight chain (i.e., unbranched) unsubstitutedalkyl group.

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond. For example, the term “alkenyl”includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl,butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.),branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups(cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, andcycloalkyl or cycloalkenyl substituted alkenyl groups. In certainembodiments, a straight chain or branched chain alkenyl group has 6 orfewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). Likewise, cycloalkenyl groups may have from3-8 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure. The term C₂-C₆ includes alkenyl groupscontaining 2 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkenyl includes both“unsubstituted alkenyls” and “substituted alkenyls,” the latter of whichrefers to alkenyl moieties having independently selected substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, alkyl groups, alkynylgroups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond. For example, the term “alkynyl”includes straight-chain alkynyl groups (e.g., ethynyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.),branched-chain alkynyl groups, and cycloalkyl or cycloalkenylsubstituted alkynyl groups. In certain embodiments, a straight chain orbranched chain alkynyl group has 6 or fewer carbon atoms in its backbone(e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain). The termC₂-C₆ includes alkynyl groups containing 2 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkynyl includes both“unsubstituted alkynyls” and “substituted alkynyls,” the latter of whichrefers to alkynyl moieties having independently selected substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, alkyl groups, alkynylgroups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withindependently selected groups such as alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulffiydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.Examples of halogen substituted alkoxy groups include, but are notlimited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,chloromethoxy, dichloromethoxy, trichloromethoxy, etc.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻(with an appropriate counterion).

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

The term “substituted” includes independently selected substituentswhich can be placed on the moiety and which allow the molecule toperform its intended function. Examples of substituents include alkyl,alkenyl, alkynyl, aryl, (CR′R″)₀₋₃NR′R″, (CR′R″)₀₋₃CN, NO₂, halogen,(CR′R″)₀₋₃C(halogen)₃, (CR′R″)₀₋₃CH(halogen)₂, (CRR″)₀₋₃CH₂(halogen),(CR′R″)₀₋₃CONR′R″, (CR′R″)₀₋₃S(O)₁₋₂NR′R″, (CR′R″)₀₋₃CHO,(CR′R″)₀₋₃O(CR′R″)₀₋₃H, (CR′R″)₀₋₃S(O)₀₋₂R′, (CR′R″)₀₋₃O(CR′R″)₀₋₃H,(CR′R″)₀₋₃COR′, (CR′R″)₀₋₃CO₂R′, or (CR′R″)₀₋₃OR′ groups; wherein eachR′ and R″ are each independently hydrogen, a C₁-C₅ alkyl, C₂-C₅ alkenyl,C₂-C₅ alkynyl, or aryl group, or R′ and R″ taken together are abenzylidene group or a —(CH₂)₂—O—(CH₂)₂— group.

The term “amine” or “amino” includes compounds or moieties in which anitrogen atom is covalently bonded to at least one carbon or heteroatom.The term “alkyl amino” includes groups and compounds wherein thenitrogen is bound to at least one additional alkyl group. The term“dialkyl amino” includes groups wherein the nitrogen atom is bound to atleast two additional alkyl groups.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl,” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “base” includes the known purine and pyrimidine heterocyclicbases, deazapurines, and analogs (including heterocyclic substitutedanalogs, e.g., aminoethyoxy phenoxazine), derivatives (e.g., 1-alkyl-,1-alkenyl-, heteroaromatic- and 1-alkynyl derivatives) and tautomersthereof. Examples of purines include adenine, guanine, inosine,diaminopurine, and xanthine and analogs (e.g., 8-oxo-N⁶-methyladenine or7-diazaxanthine) and derivatives thereof. Pyrimidines include, forexample, thymine, uracil, and cytosine, and their analogs (e.g.,5-methylcytosine, 5-methyluracil, 5-(1-propynyl)uracil,5-(1-propynyl)cytosine and 4,4-ethanocytosine). Other examples ofsuitable bases include non-purinyl and non-pyrimidinyl bases such as2-aminopyridine and triazines.

In a preferred embodiment, the nucleomonomers of an oligonucleotide ofthe invention are RNA nucleotides. In another preferred embodiment, thenucleomonomers of an oligonucleotide of the invention are modified RNAnucleotides. Thus, the oligunucleotides contain modified RNAnucleotides.

The term “nucleoside” includes bases which are covalently attached to asugar moiety, preferably ribose or deoxyribose. Examples of preferrednucleosides include ribonucleosides and deoxyribonucleosides.Nucleosides also include bases linked to amino acids or amino acidanalogs which may comprise free carboxyl groups, free amino groups, orprotecting groups. Suitable protecting groups are well known in the art(see P. G. M. Wuts and T. W. Greene, “Protective Groups in OrganicSynthesis”, 2^(nd) Ed., Wiley-Interscience, New York, 1999).

The term “nucleotide” includes nucleosides which further comprise aphosphate group or a phosphate analog.

As used herein, the term “linkage” includes a naturally occurring,unmodified phosphodiester moiety (—O—(PO²⁻)—O—) that covalently couplesadjacent nucleomonomers. As used herein, the term “substitute linkage”includes any analog or derivative of the native phosphodiester groupthat covalently couples adjacent nucleomonomers. Substitute linkagesinclude phosphodiester analogs, e.g., phosphorothioate,phosphorodithioate, and P-ethyoxyphosphodiester, P-ethoxyphosphodiester,P-alkyloxyphosphotriester, methylphosphonate, and nonphosphoruscontaining linkages, e.g., acetals and amides. Such substitute linkagesare known in the art (e.g., Bjergarde et al. 1991. Nucleic Acids Res.19:5843; Caruthers et al. 1991. Nucleosides Nucleotides. 10:47). Incertain embodiments, non-hydrolizable linkages are preferred, such asphosphorothiate linkages.

In certain embodiments, oligonucleotides of the invention comprisehydrophobicly modified nucleotides or “hydrophobic modifications.” Asused herein “hydrophobic modifications” refers to bases that aremodified such that (1) overall hydrophobicity of the base issignificantly increased, and/or (2) the base is still capable of formingclose to regular Watson—Crick interaction. Several non-limiting examplesof base modifications include 5-position uridine and cytidinemodifications such as phenyl, 4-pyridyl, 2-pyridyl, indolyl, andisobutyl, phenyl (C6H5OH); tryptophanyl (C8H6N)CH2CH(NH₂)CO), Isobutyl,butyl, aminobenzyl; phenyl; and naphthyl.

In certain embodiments, oligonucleotides of the invention comprise 3′and 5′ termini (except for circular oligonucleotides). In oneembodiment, the 3′ and 5′ termini of an oligonucleotide can besubstantially protected from nucleases e.g., by modifying the 3′ or 5′linkages (e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). For example,oligonucleotides can be made resistant by the inclusion of a “blockinggroup.” The term “blocking group” as used herein refers to substituents(e.g., other than OH groups) that can be attached to oligonucleotides ornucleomonomers, either as protecting groups or coupling groups forsynthesis (e.g., FITC, propyl (CH₂—CH₂—CH₃), glycol (—O—CH₂—CH₂—O—)phosphate (PO₃ ²), hydrogen phosphonate, or phosphoramidite). “Blockinggroups” also include “end blocking groups” or “exonuclease blockinggroups” which protect the 5′ and 3′ termini of the oligonucleotide,including modified nucleotides and non-nucleotide exonuclease resistantstructures.

Exemplary end-blocking groups include cap structures (e.g., a7-methylguanosine cap), inverted nucleomonomers, e.g., with 3′-3′ or5′-5′ end inversions (see, e.g., Ortiagao et al. 1992. Antisense Res.Dev. 2:129), methylphosphonate, phosphoramidite, non-nucleotide groups(e.g., non-nucleotide linkers, amino linkers, conjugates) and the like.The 3′ terminal nucleomonomer can comprise a modified sugar moiety. The3′ terminal nucleomonomer comprises a 3′-O that can optionally besubstituted by a blocking group that prevents 3′-exonuclease degradationof the oligonucleotide. For example, the 3′-hydroxyl can be esterifiedto a nucleotide through a 3′→3′ internucleotide linkage. For example,the alkyloxy radical can be methoxy, ethoxy, or isopropoxy, andpreferably, ethoxy. Optionally, the 3′→3′ linked nucleotide at the 3′terminus can be linked by a substitute linkage. To reduce nucleasedegradation, the 5′ most 3′→5′ linkage can be a modified linkage, e.g.,a phosphorothioate or a P-alkyloxyphosphotriester linkage. Preferably,the two 5′ most 3′→5′ linkages are modified linkages. Optionally, the 5′terminal hydroxy moiety can be esterified with a phosphorus containingmoiety, e.g., phosphate, phosphorothioate, or P-ethoxyphosphate.

Another type of conjugates that can be attached to the end (3′ or 5′end), the loop region, or any other parts of the miniRNA might include asterol, sterol type molecule, peptide, small molecule, protein, etc. Insome embodiments, a miniRNA may contain more than one conjugates (sameor different chemical nature). In some embodiments, the conjugate ischolesterol.

Another way to increase target gene specificity, or to reduce off-targetsilencing effect, is to introduce a 2′-modification (such as the 2′-Omethyl modification) at a position corresponding to the second 5′-endnucleotide of the guide sequence. This allows the positioning of this2′-modification in the Dicer-resistant hairpin structure, thus enablingone to design better RNAi constructs with less or no off-targetsilencing.

In one embodiment, a hairpin polynucleotide of the invention cancomprise one nucleic acid portion which is DNA and one nucleic acidportion which is RNA. Antisense (guide) sequences of the invention canbe “chimeric oligonucleotides” which comprise an RNA-like and a DNA-likeregion.

The language “RNase H activating region” includes a region of anoligonucleotide, e.g., a chimeric oligonucleotide, that is capable ofrecruiting RNase H to cleave the target RNA strand to which theoligonucleotide binds. Typically, the RNase activating region contains aminimal core (of at least about 3-5, typically between about 3-12, moretypically, between about 5-12, and more preferably between about 5-10contiguous nucleomonomers) of DNA or DNA-like nucleomonomers. (See,e.g., U.S. Pat. No. 5,849,902). Preferably, the RNase H activatingregion comprises about nine contiguous deoxyribose containingnucleomonomers.

The language “non-activating region” includes a region of an antisensesequence, e.g., a chimeric oligonucleotide, that does not recruit oractivate RNase H. Preferably, a non-activating region does not comprisephosphorothioate DNA. The oligonucleotides of the invention comprise atleast one non-activating region. In one embodiment, the non-activatingregion can be stabilized against nucleases or can provide specificityfor the target by being complementary to the target and forming hydrogenbonds with the target nucleic acid molecule, which is to be bound by theoligonucleotide.

In one embodiment, at least a portion of the contiguous polynucleotidesare linked by a substitute linkage, e.g., a phosphorothioate linkage.

In certain embodiments, most or all of the nucleotides beyond the guidesequence (2′-modified or not) are linked by phosphorothioate linkages.Such constructs tend to have improved pharmacokinetics due to theirhigher affinity for serum proteins. The phosphorothioate linkages in thenon-guide sequence portion of the polynucleotide generally do notinterfere with guide strand activity, once the latter is loaded intoRISC.

Antisense (guide) sequences of the present invention may include“morpholino oligonucleotides.” Morpholino oligonucleotides are non-ionicand function by an RNase H-independent mechanism. Each of the 4 geneticbases (Adenine, Cytosine, Guanine, and Thymine/Uracil) of the morpholinooligonucleotides is linked to a 6-membered morpholine ring. Morpholinooligonucleotides are made by joining the 4 different subunit types by,e.g., non-ionic phosphorodiamidate inter-subunit linkages. Morpholinooligonucleotides have many advantages including: complete resistance tonucleases (Antisense & Nucl. Acid Drug Dev. 1996. 6:267); predictabletargeting (Biochemica Biophysica Acta. 1999. 1489:141); reliableactivity in cells (Antisense & Nucl. Acid Drug Dev. 1997. 7:63);excellent sequence specificity (Antisense & Nucl. Acid Drug Dev. 1997.7:151); minimal non-antisense activity (Biochemica Biophysica Acta.1999. 1489:141); and simple osmotic or scrape delivery (Antisense &Nucl. Acid Drug Dev. 1997. 7:291). Morpholino oligonucleotides are alsopreferred because of their non-toxicity at high doses. A discussion ofthe preparation of morpholino oligonucleotides can be found in Antisense& Nucl. Acid Drug Dev. 1997. 7:187.

The chemical modifications described herein are believed, based on thedata described herein, to promote single stranded polynucleotide loadinginto the RISC. Single stranded polynucleotides have been shown to beactive in loading into RISC and inducing gene silencing. However, thelevel of activity for single stranded polynucleotides appears to be 2 to4 orders of magnitude lower when compared to a duplex polynucleotide.

The present invention provides a description of the chemicalmodification patterns, which may (a) significantly increase stability ofthe single stranded polynucleotide (b) promote efficient loading of thepolynucleotide into the RISC complex and (c) improve uptake of thesingle stranded nucleotide by the cell. FIG. 5 provides somenon-limiting examples of the chemical modification patterns which may bebeneficial for achieving single stranded polynucleotide efficacy insidethe cell. The chemical modification patterns may include combination ofribose, backbone, hydrophobic nucleoside and conjugate type ofmodifications. In addition, in some of the embodiments, the 5′ end ofthe single polynucleotide may be chemically phosphorylated.

In yet another embodiment, the present invention provides a descriptionof the chemical modifications patterns, which improve functionality ofRISC inhibiting polynucleotides. Single stranded polynucleotides havebeen shown to inhibit activity of a preloaded RISC complex through thesubstrate competition mechanism. For these types of molecules,conventionally called antagomers, the activity usually requires highconcentration and in vivo delivery is not very effective. The presentinvention provides a description of the chemical modification patterns,which may (a) significantly increase stability of the single strandedpolynucleotide (b) promote efficient recognition of the polynucleotideby the RISC as a substrate and/or (c) improve uptake of the singlestranded nucleotide by the cell. FIG. 6 provides some non-limitingexamples of the chemical modification patterns that may be beneficialfor achieving single stranded polynucleotide efficacy inside the cell.The chemical modification patterns may include combination of ribose,backbone, hydrophobic nucleoside and conjugate type of modifications.

The modifications provided by the present invention are applicable toall polynucleotides. This includes single stranded RISC enteringpolynucleotides, single stranded RISC inhibiting polynucleotides,conventional duplexed polynucleotides of variable length (15-40 bp),asymmetric duplexed polynucleotides, and the like. Polynucleotides maybe modified with wide variety of chemical modification patterns,including 5′ end, ribose, backbone and hydrophobic nucleosidemodifications.

Synthesis

Oligonucleotides of the invention can be synthesized by any method knownin the art, e.g., using enzymatic synthesis and/or chemical synthesis.The oligonucleotides can be synthesized in vitro (e.g., using enzymaticsynthesis and chemical synthesis) or in vivo (using recombinant DNAtechnology well known in the art).

In a preferred embodiment, chemical synthesis is used for modifiedpolynucleotides. Chemical synthesis of linear oligonucleotides is wellknown in the art and can be achieved by solution or solid phasetechniques. Preferably, synthesis is by solid phase methods.Oligonucleotides can be made by any of several different syntheticprocedures including the phosphoramidite, phosphite triester,H-phosphonate, and phosphotriester methods, typically by automatedsynthesis methods.

Oligonucleotide synthesis protocols are well known in the art and can befound, e.g., in U.S. Pat. No. 5,830,653; WO 98/13526; Stec et al. 1984.J. Am. Chem. Soc. 106:6077; Stec et al. 1985. J. Org. Chem. 50:3908;Stec et al. J. Chromatog. 1985. 326:263; LaPlanche et al. 1986. Nucl.Acid. Res. 1986. 14:9081; Fasman G. D., 1989. Practical Handbook ofBiochemistry and Molecular Biology. 1989. CRC Press, Boca Raton, Fla.;Lamone. 1993. Biochem. Soc. Trans. 21:1; U.S. Pat. No. 5,013,830; U.S.Pat. No. 5,214,135; U.S. Pat. No. 5,525,719; Kawasaki et al. 1993. J.Med. Chem. 36:831; WO 92/03568; U.S. Pat. No. 5,276,019; and U.S. Pat.No. 5,264,423.

The synthesis method selected can depend on the length of the desiredoligonucleotide and such choice is within the skill of the ordinaryartisan. For example, the phosphoramidite and phosphite triester methodcan produce oligonucleotides having 175 or more nucleotides, while theH-phosphonate method works well for oligonucleotides of less than 100nucleotides. If modified bases are incorporated into theoligonucleotide, and particularly if modified phosphodiester linkagesare used, then the synthetic procedures are altered as needed accordingto known procedures. In this regard, Uhlmann et al. (1990, ChemicalReviews 90:543-584) provide references and outline procedures for makingoligonucleotides with modified bases and modified phosphodiesterlinkages. Other exemplary methods for making oligonucleotides are taughtin Sonveaux. 1994. “Protecting Groups in Oligonucleotide Synthesis”;Agrawal. Methods in Molecular Biology 26:1. Exemplary synthesis methodsare also taught in “Oligonucleotide Synthesis—A Practical Approach”(Gait, M. J. IRL Press at Oxford University Press. 1984). Moreover,linear oligonucleotides of defined sequence, including some sequenceswith modified nucleotides, are readily available from several commercialsources.

The oligonucleotides may be purified by polyacrylamide gelelectrophoresis, or by any of a number of chromatographic methods,including gel chromatography and high pressure liquid chromatography. Toconfirm a nucleotide sequence, especially unmodified nucleotidesequences, oligonucleotides may be subjected to DNA sequencing by any ofthe known procedures, including Maxam and Gilbert sequencing, Sangersequencing, capillary electrophoresis sequencing, the wandering spotsequencing procedure or by using selective chemical degradation ofoligonucleotides bound to Hybond paper. Sequences of shortoligonucleotides can also be analyzed by laser desorption massspectroscopy or by fast atom bombardment (McNeal, et al., 1982, J. Am.Chem. Soc. 104:976; Viari, et al., 1987, Biomed. Environ. Mass Spectrom.14:83; Grotjahn et al., 1982, Nuc. Acid Res. 10:4671). Sequencingmethods are also available for RNA oligonucleotides.

The quality of oligonucleotides synthesized can be verified by testingthe oligonucleotide by capillary electrophoresis and denaturing stronganion HPLC(SAX-HPLC) using, e.g., the method of Bergot and Egan. 1992.J. Chrom. 599:35.

Other exemplary synthesis techniques are well known in the art (see,e.g., Sambrook et al., Molecular Cloning: a Laboratory Manual, SecondEdition (1989); DNA Cloning, Volumes I and II (D N Glover Ed. 1985);Oligonucleotide Synthesis (M J Gait Ed, 1984; Nucleic Acid Hybridisation(B D Hames and S J Higgins eds. 1984); A Practical Guide to MolecularCloning (1984); or the series, Methods in Enzymology (Academic Press,Inc.)).

In certain embodiments, the subject RNAi constructs or at least portionsthereof are transcribed from expression vectors encoding the subjectconstructs. Any art recognized vectors may be use for this purpose. Thetranscribed RNAi constructs may be isolated and purified, before desiredmodifications (such as replacing an unmodified sense strand with amodified one, etc.) are carried out.

Delivery/Carrier Uptake of Oligonucleotides by Cells

Oligonucleotides and oligonucleotide compositions are contacted with(i.e., brought into contact with, also referred to herein asadministered or delivered to) and taken up by one or more cells or acell lysate. The term “cells” includes prokaryotic and eukaryotic cells,preferably vertebrate cells, and, more preferably, mammalian cells. In apreferred embodiment, the oligonucleotide compositions of the inventionare contacted with human cells.

Oligonucleotide compositions of the invention can be contacted withcells in vitro, e.g., in a test tube or culture dish, (and may or maynot be introduced into a subject) or in vivo, e.g., in a subject such asa mammalian subject. Oligonucleotides are taken up by cells at a slowrate by endocytosis, but endocytosed oligonucleotides are generallysequestered and not available, e.g., for hybridization to a targetnucleic acid molecule. In one embodiment, cellular uptake can befacilitated by electroporation or calcium phosphate precipitation.However, these procedures are only useful for in vitro or ex vivoembodiments, are not convenient and, in some cases, are associated withcell toxicity.

In another embodiment, delivery of oligonucleotides into cells can beenhanced by suitable art recognized methods including calcium phosphate,DMSO, glycerol or dextran, electroporation, or by transfection, e.g.,using cationic, anionic, or neutral lipid compositions or liposomesusing methods known in the art (see e.g., WO 90/14074; WO 91/16024; WO91/17424; U.S. Pat. No. 4,897,355; Bergan et al. 1993. Nucleic AcidsResearch. 21:3567). Enhanced delivery of oligonucleotides can also bemediated by the use of vectors (See e.g., Shi, Y. 2003. Trends Genet.2003 Jan. 19:9; Reichhart J M et al. Genesis. 2002. 34(1-2):1604, Yu etal. 2002. Proc. Natl. Acad. Sci. USA 99:6047; Sui et al. 2002. Proc.Natl. Acad. Sci. USA 99:5515) viruses, polyamine or polycationconjugates using compounds such as polylysine, protamine, or Ni,N12-bis(ethyl) spermine (see, e.g., Bartzatt, R. et al. 1989.Biotechnol. Appl. Biochem. 11:133; Wagner E. et al. 1992. Proc. Natl.Acad. Sci. 88:4255).

In certain embodiments, the miniRNA of the invention may be delivered byusing various beta-glucan containing particles, such as those describedin US 2005/0281781 A1, WO 2006/007372, and WO 2007/050643 (allincorporated herein by reference). In certain embodiments, thebeta-glucan particle is derived from yeast. In certain embodiments, thepayload trapping molecule is a polymer, such as those with a molecularweight of at least about 1000 Da, 10,000 Da, 50,000 Da, 100 kDa, 500kDa, etc. Preferred polymers include (without limitation) cationicpolymers, chitosans, or PEI (polyethylenimine), etc.

Such beta-glucan based delivery system may be formulated for oraldelivery, where the orally delivered beta-glucan/miniRNA constructs maybe engulfed by macrophages or other related phagocytic cells, which mayin turn release the miniRNA constructs in selected in vivo sites.Alternatively or in addition, the miniRNA may changes the expression ofcertain macrophage target genes.

The optimal protocol for uptake of oligonucleotides will depend upon anumber of factors, the most crucial being the type of cells that arebeing used. Other factors that are important in uptake include, but arenot limited to, the nature and concentration of the oligonucleotide, theconfluence of the cells, the type of culture the cells are in (e.g., asuspension culture or plated) and the type of media in which the cellsare grown.

Encapsulating Agents

Encapsulating agents entrap oligonucleotides within vesicles. In anotherembodiment of the invention, an oligonucleotide may be associated with acarrier or vehicle, e.g., liposomes or micelles, although other carrierscould be used, as would be appreciated by one skilled in the art.Liposomes are vesicles made of a lipid bilayer having a structuresimilar to biological membranes. Such carriers are used to facilitatethe cellular uptake or targeting of the oligonucleotide, or improve theoligonucleotide's pharmacokinetic or toxicologic properties.

For example, the oligonucleotides of the present invention may also beadministered encapsulated in liposomes, pharmaceutical compositionswherein the active ingredient is contained either dispersed or variouslypresent in corpuscles consisting of aqueous concentric layers adherentto lipidic layers. The oligonucleotides, depending upon solubility, maybe present both in the aqueous layer and in the lipidic layer, or inwhat is generally termed a liposomic suspension. The hydrophobic layer,generally but not exclusively, comprises phopholipids such as lecithinand sphingomyelin, steroids such as cholesterol, more or less ionicsurfactants such as diacetylphosphate, stearylamine, or phosphatidicacid, or other materials of a hydrophobic nature. The diameters of theliposomes generally range from about 15 nm to about 5 microns.

The use of liposomes as drug delivery vehicles offers severaladvantages. Liposomes increase intracellular stability, increase uptakeefficiency and improve biological activity. Liposomes are hollowspherical vesicles composed of lipids arranged in a similar fashion asthose lipids which make up the cell membrane. They have an internalaqueous space for entrapping water soluble compounds and range in sizefrom 0.05 to several microns in diameter. Several studies have shownthat liposomes can deliver nucleic acids to cells and that the nucleicacids remain biologically active. For example, a lipid delivery vehicleoriginally designed as a research tool, such as Lipofectin orLIPOFECTAMINE™ 2000, can deliver intact nucleic acid molecules to cells.

Specific advantages of using liposomes include the following: they arenon-toxic and biodegradable in composition; they display longcirculation half-lives; and recognition molecules can be readilyattached to their surface for targeting to tissues. Finally,cost-effective manufacture of liposome-based pharmaceuticals, either ina liquid suspension or lyophilized product, has demonstrated theviability of this technology as an acceptable drug delivery system.

In some aspects, formulations associated with the invention might beselected for a class of naturally occurring or chemically synthesized ormodified saturated and unsaturated fatty acid residues. Fatty acidsmight exist in a form of triglycerides, diglycerides or individual fattyacids. In another embodiment, the use of well-validated mixtures offatty acids and/or fat emulsions currently used in pharmacology forparenteral nutrition may be utilized.

Liposome based formulations are widely used for oligonucleotidedelivery. However, most of commercially available lipid or liposomeformulations contain at least one positively charged lipid (cationiclipids). The presence of this positively charged lipid is believed to beessential for obtaining a high degree of oligonucleotide loading and forenhancing liposome fusogenic properties. Several methods have beenperformed and published to identify optimal positively charged lipidchemistries. However, the commercially available liposome formulationscontaining cationic lipids are characterized by a high level oftoxicity. In vivo limited therapeutic indexes have revealed thatliposome formulations containing positive charged lipids are associatedwith toxicity (i.e. elevation in liver enzymes) at concentrations onlyslightly higher than concentration required to achieve RNA silencing.

New liposome formulations, lacking the toxicity of the prior artliposomes have been developed according to the invention. These newliposome formulations are neutral fat-based formulations for theefficient delivery of oligonucleotides, and in particular for thedelivery of the RNA molecules of the invention. The compositions arereferred to as neutral nanotransporters because they enable quantitativeoligonucleotide incorporation into non-charged lipids mixtures. The lackof toxic levels of cationic lipids in the neutral nanotransportercompositions of the invention is an important feature.

The neutral nanotransporters compositions enable efficient loading ofoligonucleotide into neutral fat formulation. The composition includesan oligonucleotide that is modified in a manner such that thehydrophobicity of the molecule is increased (for example a hydrophobicmolecule is attached (covalently or no-covalently) to a hydrophobicmolecule on the oligonucleotide terminus or a non-terminal nucleotide,base, sugar, or backbone), the modified oligonucleotide being mixed witha neutral fat formulation (for example containing at least 25% ofcholesterol and 25% of DOPC or analogs thereof). A cargo molecule, suchas another lipid can also be included in the composition. Thiscomposition, where part of the formulation is build into theoligonucleotide itself, enables efficient encapsulation ofoligonucleotide in neutral lipid particles.

One of several unexpected observations associated with the invention wasthat the oligonucleotides of the invention could effectively beincorporated in a lipid mixture that was free of cationic lipids andthat such a composition could effectively deliver the therapeuticoligonucleotide to a cell in a manner that it is functional. Anotherunexpected observation was the high level of activity observed when thefatty mixture is composed of a phosphatidylcholine base fatty acid and asterol such as a cholesterol. For instance, one preferred formulation ofneutral fatty mixture is composed of at least 20% of DOPC or DSPC and atleast 20% of sterol such as cholesterol. Even as low as 1:5 lipid tooligonucleotide ratio was shown to be sufficient to get completeencapsulation of the oligonucleotide in a non charged formulation. Theprior art demonstrated only a 1-5% oligonucleotide encapsulation withnon-charged formulations, which is not sufficient to get to a desiredamount of in vivo efficacy. Compared to the prior art using neutrallipids the level of oligonucleotide delivery to a cell was quiteunexpected.

Stable particles ranging in size from 50 to 140 nm were formed uponcomplexing of hydrophobic oligonucleotides with preferred formulations.It is interesting to mention that the formulation by itself typicallydoes not form small particles, but rather, forms agglomerates, which aretransformed into stable 50-120 nm particles upon addition of thehydrophobic modified oligonucleotide.

The neutral nanotransporter compositions of the invention include ahydrophobic modified polynucleotide, a neutral fatty mixture, andoptionally a cargo molecule. A “hydrophobic modified polynucleotide” asused herein is a polynucleotide of the invention (i.e. sd-rxRNA) thathas at least one modification that renders the polynucleotide morehydrophobic than the polynucleotide was prior to modification. Themodification may be achieved by attaching (covalently or non-covalently)a hydrophobic molecule to the polynucleotide. In some instances thehydrophobic molecule is or includes a lipophilic group.

The term “lipophilic group” means a group that has a higher affinity forlipids than its affinity for water. Examples of lipophilic groupsinclude, but are not limited to, cholesterol, a cholesteryl or modifiedcholesteryl residue, adamantine, dihydrotesterone, long chain alkyl,long chain alkenyl, long chain alkynyl, olely-lithocholic, cholenic,oleoyl-cholenic, palmityl, heptadecyl, myrisityl, bile acids, cholicacid or taurocholic acid, deoxycholate, oleyl litocholic acid, oleoylcholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids,such as steroids, vitamins, such as vitamin E, fatty acids eithersaturated or unsaturated, fatty acid esters, such as triglycerides,pyrenes, porphyrines, Texaphyrine, adamantane, acridines, biotin,coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin,dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyaninedyes (e.g. Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen. Thecholesterol moiety may be reduced (e.g. as in cholestan) or may besubstituted (e.g. by halogen). A combination of different lipophilicgroups in one molecule is also possible.

The hydrophobic molecule may be attached at various positions of thepolynucleotide. As described above, the hydrophobic molecule may belinked to the terminal residue of the polynucleotide such as the 3′ of5′-end of the polynucleotide. Alternatively, it may be linked to aninternal nucleotide or a nucleotide on a branch of the polynucleotide.The hydrophobic molecule may be attached, for instance to a 2′-positionof the nucleotide. The hydrophobic molecule may also be linked to theheterocyclic base, the sugar or the backbone of a nucleotide of thepolynucleotide.

The hydrophobic molecule may be connected to the polynucleotide by alinker moiety. Optionally the linker moiety is a non-nucleotidic linkermoiety. Non-nucleotidic linkers are e.g. abasic residues (dSpacer),oligoethyleneglycol, such as triethyleneglycol (spacer 9) orhexaethylenegylcol (spacer 18), or alkane-diol, such as butanediol. Thespacer units are preferably linked by phosphodiester or phosphorothioatebonds. The linker units may appear just once in the molecule or may beincorporated several times, e.g. via phosphodiester, phosphorothioate,methylphosphonate, or amide linkages.

Typical conjugation protocols involve the synthesis of polynucleotidesbearing an aminolinker at one or more positions of the sequence,however, a linker is not required. The amino group is then reacted withthe molecule being conjugated using appropriate coupling or activatingreagents. The conjugation reaction may be performed either with thepolynucleotide still bound to a solid support or following cleavage ofthe polynucleotide in solution phase. Purification of the modifiedpolynucleotide by HPLC typically results in a pure material.

In some embodiments the hydrophobic molecule is a sterol type conjugate,a PhytoSterol conjugate, cholesterol conjugate, sterol type conjugatewith altered side chain length, fatty acid conjugate, any otherhydrophobic group conjugate, and/or hydrophobic modifications of theinternal nucleoside, which provide sufficient hydrophobicity to beincorporated into micelles.

For purposes of the present invention, the term “sterols”, refers orsteroid alcohols are a subgroup of steroids with a hydroxyl group at the3-position of the A-ring. They are amphipathic lipids synthesized fromacetyl-coenzyme A via the HMG-CoA reductase pathway. The overallmolecule is quite flat. The hydroxyl group on the A ring is polar. Therest of the aliphatic chain is non-polar. Usually sterols are consideredto have an 8 carbon chain at position 17.

For purposes of the present invention, the term “sterol type molecules”,refers to steroid alcohols, which are similar in structure to sterols.The main difference is the structure of the ring and number of carbonsin a position 21 attached side chain.

For purposes of the present invention, the term “PhytoSterols” (alsocalled plant sterols) are a group of steroid alcohols, phytochemicalsnaturally occurring in plants. There are more then 200 different knownPhytoSterols

For purposes of the present invention, the term “Sterol side chain”refers to a chemical composition of a side chain attached at theposition 17 of sterol-type molecule. In a standard definition sterolsare limited to a 4 ring structure carrying a 8 carbon chain at position17. In this invention, the sterol type molecules with side chain longerand shorter than conventional are described. The side chain may branchedor contain double back bones.

Thus, sterols useful in the invention, for example, includecholesterols, as well as unique sterols in which position 17 hasattached side chain of 2-7 or longer then 9 carbons. In a particularembodiment, the length of the polycarbon tail is varied between 5 and 9carbons. FIG. 9 demonstrates that there is a correlation between plasmaclearance, liver uptake and the length of the polycarbon chain. Suchconjugates may have significantly better in vivo efficacy, in particulardelivery to liver. These types of molecules are expected to work atconcentrations 5 to 9 fold lower then oligonucleotides conjugated toconventional cholesterols.

Alternatively the polynucleotide may be bound to a protein, peptide orpositively charged chemical that functions as the hydrophobic molecule.The proteins may be selected from the group consisting of protamine,dsRNA binding domain, and arginine rich peptides. Exemplary positivelycharged chemicals include spermine, spermidine, cadaverine, andputrescine.

In another embodiment hydrophobic molecule conjugates may demonstrateeven higher efficacy when it is combined with optimal chemicalmodification patterns of the polynucleotide (as described herein indetail), containing but not limited to hydrophobic modifications,phosphorothioate modifications, and 2′ ribo modifications.

In another embodiment the sterol type molecule may be a naturallyoccurring PhytoSterols such as those shown in FIG. 8. The polycarbonchain may be longer than 9 and may be linear, branched and/or containdouble bonds. Some PhytoSterol containing polynucleotide conjugates maybe significantly more potent and active in delivery of polynucleotidesto various tissues. Some PhytoSterols may demonstrate tissue preferenceand thus be used as a way to delivery RNAi specifically to particulartissues.

The hydrophobic modified polynucleotide is mixed with a neutral fattymixture to form a micelle. The neutral fatty acid mixture is a mixtureof fats that has a net neutral or slightly net negative charge at oraround physiological pH that can form a micelle with the hydrophobicmodified polynucleotide. For purposes of the present invention, the term“micelle” refers to a small nanoparticle formed by a mixture of noncharged fatty acids and phospholipids. The neutral fatty mixture mayinclude cationic lipids as long as they are present in an amount thatdoes not cause toxicity. In preferred embodiments the neutral fattymixture is free of cationic lipids. A mixture that is free of cationiclipids is one that has less than 1% and preferably 0% of the total lipidbeing cationic lipid. The term “cationic lipid” includes lipids andsynthetic lipids having a net positive charge at or around physiologicalpH. The term “anionic lipid” includes lipids and synthetic lipids havinga net negative charge at or around physiological pH.

The neutral fats bind to the oligonucleotides of the invention by astrong but non-covalent attraction (e.g., an electrostatic, van derWaals, pi-stacking, etc. interaction).

The neutral fat mixture may include formulations selected from a classof naturally occurring or chemically synthesized or modified saturatedand unsaturated fatty acid residues. Fatty acids might exist in a formof triglycerides, diglycerides or individual fatty acids. In anotherembodiment the use of well-validated mixtures of fatty acids and/or fatemulsions currently used in pharmacology for parenteral nutrition may beutilized.

The neutral fatty mixture is preferably a mixture of a choline basedfatty acid and a sterol. Choline based fatty acids include for instance,synthetic phosphocholine derivatives such as DDPC, DLPC, DMPC, DPPC,DSPC, DOPC, POPC, and DEPC. DOPC (chemical registry number 4235-95-4) isdioleoylphosphatidylcholine (also known asdielaidoylphosphatidylcholine, dioleoyl-PC, dioleoylphosphocholine,dioleoyl-sn-glycero-3-phosphocholine, dioleylphosphatidylcholine). DSPC(chemical registry number 816-94-4) is distearoylphosphatidylcholine(also known as 1,2-Distearoyl-sn-Glycero-3-phosphocholine).

The sterol in the neutral fatty mixture may be for instance cholesterol.The neutral fatty mixture may be made up completely of a choline basedfatty acid and a sterol or it may optionally include a cargo molecule.For instance, the neutral fatty mixture may have at least 20% or 25%fatty acid and 20% or 25% sterol.

For purposes of the present invention, the term “Fatty acids” relates toconventional description of fatty acid. They may exist as individualentities or in a form of two- and triglycerides. For purposes of thepresent invention, the term “fat emulsions” refers to safe fatformulations given intravenously to subjects who are unable to getenough fat in their diet. It is an emulsion of soy bean oil (or othernaturally occurring oils) and egg phospholipids. Fat emulsions are beingused for formulation of some insoluble anesthetics. In this disclosure,fat emulsions might be part of commercially available preparations likeIntralipid, Liposyn, Nutrilipid, modified commercial preparations, wherethey are enriched with particular fatty acids or fully denovo-formulated combinations of fatty acids and phospholipids.

In one embodiment, the cells to be contacted with an oligonucleotidecomposition of the invention are contacted with a mixture comprising theoligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 12 hoursto about 24 hours. In another embodiment, the cells to be contacted withan oligonucleotide composition are contacted with a mixture comprisingthe oligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 1 andabout five days. In one embodiment, the cells are contacted with amixture comprising a lipid and the oligonucleotide for between aboutthree days to as long as about 30 days. In another embodiment, a mixturecomprising a lipid is left in contact with the cells for at least aboutfive to about 20 days. In another embodiment, a mixture comprising alipid is left in contact with the cells for at least about seven toabout 15 days.

50%-60% of the formulation can optionally be any other lipid ormolecule. Such a lipid or molecule is referred to herein as a cargolipid or cargo molecule. Cargo molecules include but are not limited tointralipid, small molecules, fusogenic peptides or lipids or other smallmolecules might be added to alter cellular uptake, endosomal release ortissue distribution properties. The ability to tolerate cargo moleculesis important for modulation of properties of these particles, if suchproperties are desirable. For instance the presence of some tissuespecific metabolites might drastically alter tissue distributionprofiles. For example use of Intralipid type formulation enriched inshorter or longer fatty chains with various degrees of saturationaffects tissue distribution profiles of these type of formulations (andtheir loads).

An example of a cargo lipid useful according to the invention is afusogenic lipid. For instance, the zwiterionic lipid DOPE (chemicalregistry number 4004-5-1,1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine)is a preferred cargo lipid.

Intralipid may be comprised of the following composition: 1 000 mLcontain: purified soybean oil 90 g, purified egg phospholipids 12 g,glycerol anhydrous 22 g, water for injection q.s. ad 1 000 mL. pH isadjusted with sodium hydroxide to pH approximately 8. Energy content/L:4.6 MJ (190 kcal). Osmolality (approx.): 300 mOsm/kg water. In anotherembodiment fat emulsion is Liposyn that contains 5% safflower oil, 5%soybean oil, up to 1.2% egg phosphatides added as an emulsifier and 2.5%glycerin in water for injection. It may also contain sodium hydroxidefor pH adjustment. pH 8.0 (6.0-9.0). Liposyn has an osmolarity of 276 mOsmol/liter (actual). Variation in the identity, amounts and ratios ofcargo lipids affects the cellular uptake and tissue distributioncharacteristics of these compounds. For example, the length of lipidtails and level of saturability will affect differential uptake toliver, lung, fat and cardiomyocytes. Addition of special hydrophobicmolecules like vitamins or different forms of sterols can favordistribution to special tissues which are involved in the metabolism ofparticular compounds. Complexes are formed at different oligonucleotideconcentrations, with higher concentrations favoring more efficientcomplex formation (FIGS. 21-22).

In another embodiment, the fat emulsion is based on a mixture of lipids.Such lipids may include natural compounds, chemically synthesizedcompounds, purified fatty acids or any other lipids. In yet anotherembodiment the composition of fat emulsion is entirely artificial. In aparticular embodiment, the fat emulsion is more then 70% linoleic acid.In yet another particular embodiment the fat emulsion is at least 1% ofcardiolipin. Linoleic acid (LA) is an unsaturated omega-6 fatty acid. Itis a colorless liquid made of a carboxylic acid with an 18-carbon chainand two cis double bonds.

In yet another embodiment of the present invention, the alteration ofthe composition of the fat emulsion is used as a way to alter tissuedistribution of hydrophobicly modified polynucleotides. This methodologyprovides for the specific delivery of the polynucleotides to particulartissues (FIG. 12).

In another embodiment the fat emulsions of the cargo molecule containmore then 70% of Linoleic acid (C18H32O2) and/or cardiolipin are usedfor specifically delivering RNAi to heart muscle.

Fat emulsions, like intralipid have been used before as a deliveryformulation for some non-water soluble drugs (such as Propofol,re-formulated as Diprivan). Unique features of the present inventioninclude (a) the concept of combining modified polynucleotides with thehydrophobic compound(s), so it can be incorporated in the fat micellesand (b) mixing it with the fat emulsions to provide a reversiblecarrier. After injection into a blood stream, micelles usually bind toserum proteins, including albumin, HDL, LDL and other. This binding isreversible and eventually the fat is absorbed by cells. Thepolynucleotide, incorporated as a part of the micelle will then bedelivered closely to the surface of the cells. After that cellularuptake might be happening though variable mechanisms, including but notlimited to sterol type delivery.

Complexing Agents

Complexing agents bind to the oligonucleotides of the invention by astrong but non-covalent attraction (e.g., an electrostatic, van derWaals, pi-stacking, etc. interaction). In one embodiment,oligonucleotides of the invention can be complexed with a complexingagent to increase cellular uptake of oligonucleotides. An example of acomplexing agent includes cationic lipids. Cationic lipids can be usedto deliver oligonucleotides to cells. However, as discussed above,formulations free in cationic lipids are preferred in some embodiments.

The term “cationic lipid” includes lipids and synthetic lipids havingboth polar and non-polar domains and which are capable of beingpositively charged at or around physiological pH and which bind topolyanions, such as nucleic acids, and facilitate the delivery ofnucleic acids into cells. In general cationic lipids include saturatedand unsaturated alkyl and alicyclic ethers and esters of amines, amides,or derivatives thereof. Straight-chain and branched alkyl and alkenylgroups of cationic lipids can contain, e.g., from 1 to about 25 carbonatoms. Preferred straight chain or branched alkyl or alkene groups havesix or more carbon atoms. Alicyclic groups include cholesterol and othersteroid groups. Cationic lipids can be prepared with a variety ofcounterions (anions) including, e.g., Cl⁻, Br⁻, I⁻, F⁻, acetate,trifluoroacetate, sulfate, nitrite, and nitrate.

Examples of cationic lipids include polyethylenimine, polyamidoamine(PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA andDOPE), Lipofectase, LIPOFECTAMINE™ (e.g., LIPOFECTAMINE™ 2000), DOPE,Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL,San Luis Obispo, Calif.). Exemplary cationic liposomes can be made fromN-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA),N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate(DOTAP), 3β-[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol(DC-Chol),2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; anddimethyldioctadecylammonium bromide (DDAB). The cationic lipidN-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),for example, was found to increase 1000-fold the antisense effect of aphosphorothioate oligonucleotide. (Vlassov et al., 1994, Biochimica etBiophysica Acta 1197:95-108). Oligonucleotides can also be complexedwith, e.g., poly(L-lysine) or avidin and lipids may, or may not, beincluded in this mixture, e.g., steryl-poly(L-lysine).

Cationic lipids have been used in the art to deliver oligonucleotides tocells (see, e.g., U.S. Pat. Nos. 5,855,910; 5,851,548; 5,830,430;5,780,053; 5,767,099; Lewis et al. 1996. Proc. Natl. Acad. Sci. USA93:3176; Hope et al. 1998. Molecular Membrane Biology 15:1). Other lipidcompositions which can be used to facilitate uptake of the instantoligonucleotides can be used in connection with the claimed methods. Inaddition to those listed supra, other lipid compositions are also knownin the art and include, e.g., those taught in U.S. Pat. No. 4,235,871;U.S. Pat. Nos. 4,501,728; 4,837,028; 4,737,323.

In one embodiment lipid compositions can further comprise agents, e.g.,viral proteins to enhance lipid-mediated transfections ofoligonucleotides (Kamata, et al., 1994. Nucl. Acids. Res. 22:536). Inanother embodiment, oligonucleotides are contacted with cells as part ofa composition comprising an oligonucleotide, a peptide, and a lipid astaught, e.g., in U.S. Pat. No. 5,736,392. Improved lipids have also beendescribed which are serum resistant (Lewis, et al., 1996. Proc. Natl.Acad. Sci. 93:3176). Cationic lipids and other complexing agents act toincrease the number of oligonucleotides carried into the cell throughendocytosis.

In another embodiment N-substituted glycine oligonucleotides (peptoids)can be used to optimize uptake of oligonucleotides. Peptoids have beenused to create cationic lipid-like compounds for transfection (Murphy,et al., 1998. Proc. Natl. Acad. Sci. 95:1517). Peptoids can besynthesized using standard methods (e.g., Zuckermann, R. N., et al.1992. J. Am. Chem. Soc. 114:10646; Zuckermann, R. N., et al. 1992. Int.J. Peptide Protein Res. 40:497). Combinations of cationic lipids andpeptoids, liptoids, can also be used to optimize uptake of the subjectoligonucleotides (Hunag, et al., 1998. Chemistry and Biology. 5:345).Liptoids can be synthesized by elaborating peptoid oligonucleotides andcoupling the amino terminal submonomer to a lipid via its amino group(Hunag, et al., 1998. Chemistry and Biology. 5:345).

It is known in the art that positively charged amino acids can be usedfor creating highly active cationic lipids (Lewis et al. 1996. Proc.Natl. Acad. Sci. U.S.A. 93:3176). In one embodiment, a composition fordelivering oligonucleotides of the invention comprises a number ofarginine, lysine, histidine or ornithine residues linked to a lipophilicmoiety (see e.g., U.S. Pat. No. 5,777,153).

In another embodiment, a composition for delivering oligonucleotides ofthe invention comprises a peptide having from between about one to aboutfour basic residues. These basic residues can be located, e.g., on theamino terminal, C-terminal, or internal region of the peptide. Familiesof amino acid residues having similar side chains have been defined inthe art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine (canalso be considered non-polar), asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Apart from the basic amino acids, a majority or all of theother residues of the peptide can be selected from the non-basic aminoacids, e.g., amino acids other than lysine, arginine, or histidine.Preferably a preponderance of neutral amino acids with long neutral sidechains are used.

In one embodiment, a composition for delivering oligonucleotides of theinvention comprises a natural or synthetic polypeptide having one ormore gamma carboxyglutamic acid residues, or γ-Gla residues. These gammacarboxyglutamic acid residues may enable the polypeptide to bind to eachother and to membrane surfaces. In other words, a polypeptide having aseries of γ-Gla may be used as a general delivery modality that helps anRNAi construct to stick to whatever membrane to which it comes incontact. This may at least slow RNAi constructs from being cleared fromthe blood stream and enhance their chance of homing to the target.

The gamma carboxyglutamic acid residues may exist in natural proteins(for example, prothrombin has 10 γ-Gla residues). Alternatively, theycan be introduced into the purified, recombinantly produced, orchemically synthesized polypeptides by carboxylation using, for example,a vitamin K-dependent carboxylase. The gamma carboxyglutamic acidresidues may be consecutive or non-consecutive, and the total number andlocation of such gamma carboxyglutamic acid residues in the polypeptidecan be regulated/fine tuned to achieve different levels of “stickiness”of the polypeptide.

In one embodiment, the cells to be contacted with an oligonucleotidecomposition of the invention are contacted with a mixture comprising theoligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 12 hoursto about 24 hours. In another embodiment, the cells to be contacted withan oligonucleotide composition are contacted with a mixture comprisingthe oligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 1 andabout five days. In one embodiment, the cells are contacted with amixture comprising a lipid and the oligonucleotide for between aboutthree days to as long as about 30 days. In another embodiment, a mixturecomprising a lipid is left in contact with the cells for at least aboutfive to about 20 days. In another embodiment, a mixture comprising alipid is left in contact with the cells for at least about seven toabout 15 days.

For example, in one embodiment, an oligonucleotide composition can becontacted with cells in the presence of a lipid such as cytofectin CS orGSV (available from Glen Research; Sterling, Va.), GS3815, GS2888 forprolonged incubation periods as described herein.

In one embodiment, the incubation of the cells with the mixturecomprising a lipid and an oligonucleotide composition does not reducethe viability of the cells. Preferably, after the transfection periodthe cells are substantially viable. In one embodiment, aftertransfection, the cells are between at least about 70% and at leastabout 100% viable. In another embodiment, the cells are between at leastabout 80% and at least about 95% viable. In yet another embodiment, thecells are between at least about 85% and at least about 90% viable.

In one embodiment, oligonucleotides are modified by attaching a peptidesequence that transports the oligonucleotide into a cell, referred toherein as a “transporting peptide.” In one embodiment, the compositionincludes an oligonucleotide which is complementary to a target nucleicacid molecule encoding the protein, and a covalently attachedtransporting peptide.

The language “transporting peptide” includes an amino acid sequence thatfacilitates the transport of an oligonucleotide into a cell. Exemplarypeptides which facilitate the transport of the moieties to which theyare linked into cells are known in the art, and include, e.g., HIV TATtranscription factor, lactoferrin, Herpes VP22 protein, and fibroblastgrowth factor 2 (Pooga et al. 1998. Nature Biotechnology. 16:857; andDerossi et al. 1998. Trends in Cell Biology. 8:84; Elliott and O'Hare.1997. Cell 88:223).

Oligonucleotides can be attached to the transporting peptide using knowntechniques, e.g., (Prochiantz, A. 1996. Curr. Opin. Neurobiol. 6:629;Derossi et al. 1998. Trends Cell Biol. 8:84; Troy et al. 1996. J.Neurosci. 16:253), Vives et al. 1997. J. Biol. Chem. 272:16010). Forexample, in one embodiment, oligonucleotides bearing an activated thiolgroup are linked via that thiol group to a cysteine present in atransport peptide (e.g., to the cysteine present in the (3 turn betweenthe second and the third helix of the antennapedia homeodomain astaught, e.g., in Derossi et al. 1998. Trends Cell Biol. 8:84;Prochiantz. 1996. Current Opinion in Neurobiol. 6:629; Allinquant et al.1995. J. Cell Biol. 128:919). In another embodiment, a Boc-Cys-(Npys)OHgroup can be coupled to the transport peptide as the last (N-terminal)amino acid and an oligonucleotide bearing an SH group can be coupled tothe peptide (Troy et al. 1996. J. Neurosci. 16:253).

In one embodiment, a linking group can be attached to a nucleomonomerand the transporting peptide can be covalently attached to the linker.In one embodiment, a linker can function as both an attachment site fora transporting peptide and can provide stability against nucleases.Examples of suitable linkers include substituted or unsubstituted C₁-C₂₀alkyl chains, C₂-C₂₀ alkenyl chains, C₂-C₂₀ alkynyl chains, peptides,and heteroatoms (e.g., S, O, NH, etc.). Other exemplary linkers includebifunctional crosslinking agents such assulfosuccinimidyl-4-(maleimidophenyl)-butyrate (SMPB) (see, e.g., Smithet al. Biochem J 1991.276: 417-2).

In one embodiment, oligonucleotides of the invention are synthesized asmolecular conjugates which utilize receptor-mediated endocytoticmechanisms for delivering genes into cells (see, e.g., Bunnell et al.1992. Somatic Cell and Molecular Genetics. 18:559, and the referencescited therein).

Targeting Agents

The delivery of oligonucleotides can also be improved by targeting theoligonucleotides to a cellular receptor. The targeting moieties can beconjugated to the oligonucleotides or attached to a carrier group (i.e.,poly(L-lysine) or liposomes) linked to the oligonucleotides. This methodis well suited to cells that display specific receptor-mediatedendocytosis.

For instance, oligonucleotide conjugates to 6-phosphomannosylatedproteins are internalized 20-fold more efficiently by cells expressingmannose 6-phosphate specific receptors than free oligonucleotides. Theoligonucleotides may also be coupled to a ligand for a cellular receptorusing a biodegradable linker. In another example, the delivery constructis mannosylated streptavidin which forms a tight complex withbiotinylated oligonucleotides. Mannosylated streptavidin was found toincrease 20-fold the internalization of biotinylated oligonucleotides.(Vlassov et al. 1994. Biochimica et Biophysica Acta 1197:95-108).

In addition specific ligands can be conjugated to the polylysinecomponent of polylysine-based delivery systems. For example,transferrin-polylysine, adenovirus-polylysine, and influenza virushemagglutinin HA-2 N-terminal fusogenic peptides-polylysine conjugatesgreatly enhance receptor-mediated DNA delivery in eucaryotic cells.Mannosylated glycoprotein conjugated to poly(L-lysine) in aveolarmacrophages has been employed to enhance the cellular uptake ofoligonucleotides. Liang et al. 1999. Pharmazie 54:559-566.

Because malignant cells have an increased need for essential nutrientssuch as folic acid and transferrin, these nutrients can be used totarget oligonucleotides to cancerous cells. For example, when folic acidis linked to poly(L-lysine) enhanced oligonucleotide uptake is seen inpromyelocytic leukaemia (HL-60) cells and human melanoma (M−14) cells.Ginobbi et al. 1997. Anticancer Res. 17:29. In another example,liposomes coated with maleylated bovine serum albumin, folic acid, orferric protoporphyrin IX, show enhanced cellular uptake ofoligonucleotides in murine macrophages, KB cells, and 2.2.15 humanhepatoma cells. Liang et al. 1999. Pharmazie 54:559-566.

Liposomes naturally accumulate in the liver, spleen, andreticuloendothelial system (so-called, passive targeting). By couplingliposomes to various ligands such as antibodies are protein A, they canbe actively targeted to specific cell populations. For example, proteinA-bearing liposomes may be pretreated with H-2K specific antibodieswhich are targeted to the mouse major histocompatibility complex-encodedH-2K protein expressed on L cells. (Vlassov et al. 1994. Biochimica etBiophysica Acta 1197:95-108).

Other in vitro and/or in vivo delivery of RNAi reagents are known in theart, and can be used to deliver the subject RNAi constructs. See, forexample, U.S. patent application publications 20080152661, 20080112916,20080107694, 20080038296, 20070231392, 20060240093, 20060178327,20060008910, 20050265957, 20050064595, 20050042227, 20050037496,20050026286, 20040162235, 20040072785, 20040063654, 20030157030, WO2008/036825, WO04/065601, and AU2004206255B2, just to name a few (allincorporated by reference).

Administration

The optimal course of administration or delivery of the oligonucleotidesmay vary depending upon the desired result and/or on the subject to betreated. As used herein “administration” refers to contacting cells witholigonucleotides and can be performed in vitro or in vivo. The dosage ofoligonucleotides may be adjusted to optimally reduce expression of aprotein translated from a target nucleic acid molecule, e.g., asmeasured by a readout of RNA stability or by a therapeutic response,without undue experimentation.

For example, expression of the protein encoded by the nucleic acidtarget can be measured to determine whether or not the dosage regimenneeds to be adjusted accordingly. In addition, an increase or decreasein RNA or protein levels in a cell or produced by a cell can be measuredusing any art recognized technique. By determining whether transcriptionhas been decreased, the effectiveness of the oligonucleotide in inducingthe cleavage of a target RNA can be determined.

Any of the above-described oligonucleotide compositions can be usedalone or in conjunction with a pharmaceutically acceptable carrier. Asused herein, “pharmaceutically acceptable carrier” includes appropriatesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutical active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, it can be used in thetherapeutic compositions. Supplementary active ingredients can also beincorporated into the compositions.

Oligonucleotides may be incorporated into liposomes or liposomesmodified with polyethylene glycol or admixed with cationic lipids forparenteral administration. Incorporation of additional substances intothe liposome, for example, antibodies reactive against membrane proteinsfound on specific target cells, can help target the oligonucleotides tospecific cell types.

Moreover, the present invention provides for administering the subjectoligonucleotides with an osmotic pump providing continuous infusion ofsuch oligonucleotides, for example, as described in Rataiczak et al.(1992 Proc. Natl. Acad. Sci. USA 89:11823-11827). Such osmotic pumps arecommercially available, e.g., from Alzet Inc. (Palo Alto, Calif.).Topical administration and parenteral administration in a cationic lipidcarrier are preferred.

With respect to in vivo applications, the formulations of the presentinvention can be administered to a patient in a variety of forms adaptedto the chosen route of administration, e.g., parenterally, orally, orintraperitoneally. Parenteral administration, which is preferred,includes administration by the following routes: intravenous;intramuscular; interstitially; intraarterially; subcutaneous; intraocular; intrasynovial; trans epithelial, including transdermal;pulmonary via inhalation; ophthalmic; sublingual and buccal; topically,including ophthalmic; dermal; ocular; rectal; and nasal inhalation viainsufflation.

Pharmaceutical preparations for parenteral administration includeaqueous solutions of the active compounds in water-soluble orwater-dispersible form. In addition, suspensions of the active compoundsas appropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension include, for example,sodium carboxymethyl cellulose, sorbitol, or dextran, optionally, thesuspension may also contain stabilizers. The oligonucleotides of theinvention can be formulated in liquid solutions, preferably inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the oligonucleotides may be formulated in solidform and redissolved or suspended immediately prior to use. Lyophilizedforms are also included in the invention.

Pharmaceutical preparations for topical administration includetransdermal patches, ointments, lotions, creams, gels, drops, sprays,suppositories, liquids and powders. In addition, conventionalpharmaceutical carriers, aqueous, powder or oily bases, or thickenersmay be used in pharmaceutical preparations for topical administration.

Pharmaceutical preparations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. In addition, thickeners, flavoring agents,diluents, emulsifiers, dispersing aids, or binders may be used inpharmaceutical preparations for oral administration.

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are known in the art, and include, for example, fortransmucosal administration bile salts and fusidic acid derivatives, anddetergents. Transmucosal administration may be through nasal sprays orusing suppositories. For oral administration, the oligonucleotides areformulated into conventional oral administration forms such as capsules,tablets, and tonics. For topical administration, the oligonucleotides ofthe invention are formulated into ointments, salves, gels, or creams asknown in the art.

Drug delivery vehicles can be chosen e.g., for in vitro, for systemic,or for topical administration. These vehicles can be designed to serveas a slow release reservoir or to deliver their contents directly to thetarget cell. An advantage of using some direct delivery drug vehicles isthat multiple molecules are delivered per uptake. Such vehicles havebeen shown to increase the circulation half-life of drugs that wouldotherwise be rapidly cleared from the blood stream. Some examples ofsuch specialized drug delivery vehicles which fall into this categoryare liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres.

The described oligonucleotides may be administered systemically to asubject. Systemic absorption refers to the entry of drugs into the bloodstream followed by distribution throughout the entire body.Administration routes which lead to systemic absorption include:intravenous, subcutaneous, intraperitoneal, and intranasal. Each ofthese administration routes delivers the oligonucleotide to accessiblediseased cells. Following subcutaneous administration, the therapeuticagent drains into local lymph nodes and proceeds through the lymphaticnetwork into the circulation. The rate of entry into the circulation hasbeen shown to be a function of molecular weight or size. The use of aliposome or other drug carrier localizes the oligonucleotide at thelymph node. The oligonucleotide can be modified to diffuse into thecell, or the liposome can directly participate in the delivery of eitherthe unmodified or modified oligonucleotide into the cell.

The chosen method of delivery will result in entry into cells. Preferreddelivery methods include liposomes (10-400 nm), hydrogels,controlled-release polymers, and other pharmaceutically applicablevehicles, and microinjection or electroporation (for ex vivotreatments).

The pharmaceutical preparations of the present invention may be preparedand formulated as emulsions. Emulsions are usually heterogeneous systemsof one liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter. The emulsions of the present invention maycontain excipients such as emulsifiers, stabilizers, dyes, fats, oils,waxes, fatty acids, fatty alcohols, fatty esters, humectants,hydrophilic colloids, preservatives, and anti-oxidants may also bepresent in emulsions as needed. These excipients may be present as asolution in either the aqueous phase, oily phase or itself as a separatephase.

Examples of naturally occurring emulsifiers that may be used in emulsionformulations of the present invention include lanolin, beeswax,phosphatides, lecithin and acacia. Finely divided solids have also beenused as good emulsifiers especially in combination with surfactants andin viscous preparations. Examples of finely divided solids that may beused as emulsifiers include polar inorganic solids, such as heavy metalhydroxides, nonswelling clays such as bentonite, attapulgite, hectorite,kaolin, montrnorillonite, colloidal aluminum silicate and colloidalmagnesium aluminum silicate, pigments and nonpolar solids such as carbonor glyceryl tristearate.

Examples of preservatives that may be included in the emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Examples of antioxidants that may be included in the emulsionformulations include free radical scavengers such as tocopherols, alkylgallates, butylated hydroxyanisole, butylated hydroxytoluene, orreducing agents such as ascorbic acid and sodium metabisulfite, andantioxidant synergists such as citric acid, tartaric acid, and lecithin.

In one embodiment, the compositions of oligonucleotides are formulatedas microemulsions. A microemulsion is a system of water, oil andamphiphile which is a single optically isotropic and thermodynamicallystable liquid solution. Typically microemulsions are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a 4th component, generally an intermediatechain-length alcohol to form a transparent system.

Surfactants that may be used in the preparation of microemulsionsinclude, but are not limited to, ionic surfactants, non-ionicsurfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fattyacid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate(MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate(PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate(MO750), decaglycerol sequioleate (S0750), decaglycerol decaoleate(DA0750), alone or in combination with cosurfactants. The cosurfactant,usually a short-chain alcohol such as ethanol, 1-propanol, and1-butanol, serves to increase the interfacial fluidity by penetratinginto the surfactant film and consequently creating a disordered filmbecause of the void space generated among surfactant molecules.

Microemulsions may, however, be prepared without the use ofcosurfactants and alcohol-free self-emulsifying microemulsion systemsare known in the art. The aqueous phase may typically be, but is notlimited to, water, an aqueous solution of the drug, glycerol, PEG300,PEG400, polyglycerols, propylene glycols, and derivatives of ethyleneglycol. The oil phase may include, but is not limited to, materials suchas Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain(C₈-C₁₂) mono, di, and tri-glycerides, polyoxyethylated glyceryl fattyacid esters, fatty alcohols, polyglycolized glycerides, saturatedpolyglycolized C₈-C₁₀ glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both oil/water and water/oil) have been proposed toenhance the oral bioavailability of drugs.

Microemulsions offer improved drug solubilization, protection of drugfrom enzymatic hydrolysis, possible enhancement of drug absorption dueto surfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11:1385; Ho et al., J. Pharm.Sci., 1996, 85:138-143). Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides from thegastrointestinal tract, as well as improve the local cellular uptake ofoligonucleotides within the gastrointestinal tract, vagina, buccalcavity and other areas of administration.

In an embodiment, the present invention employs various penetrationenhancers to affect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Evennon-lipophilic drugs may cross cell membranes if the membrane to becrossed is treated with a penetration enhancer. In addition toincreasing the diffusion of non-lipophilic drugs across cell membranes,penetration enhancers also act to enhance the permeability of lipophilicdrugs.

Five categories of penetration enhancers that may be used in the presentinvention include: surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants. Other agents may be utilizedto enhance the penetration of the administered oligonucleotides include:glycols such as ethylene glycol and propylene glycol, pyrrols such as2-15 pyrrol, azones, and terpenes such as limonene, and menthone.

The oligonucleotides, especially in lipid formulations, can also beadministered by coating a medical device, for example, a catheter, suchas an angioplasty balloon catheter, with a cationic lipid formulation.Coating may be achieved, for example, by dipping the medical device intoa lipid formulation or a mixture of a lipid formulation and a suitablesolvent, for example, an aqueous-based buffer, an aqueous solvent,ethanol, methylene chloride, chloroform and the like. An amount of theformulation will naturally adhere to the surface of the device which issubsequently administered to a patient, as appropriate. Alternatively, alyophilized mixture of a lipid formulation may be specifically bound tothe surface of the device. Such binding techniques are described, forexample, in K. Ishihara et al., Journal of Biomedical MaterialsResearch, Vol. 27, pp. 1309-1314 (1993), the disclosures of which areincorporated herein by reference in their entirety.

The useful dosage to be administered and the particular mode ofadministration will vary depending upon such factors as the cell type,or for in vivo use, the age, weight and the particular animal and regionthereof to be treated, the particular oligonucleotide and deliverymethod used, the therapeutic or diagnostic use contemplated, and theform of the formulation, for example, suspension, emulsion, micelle orliposome, as will be readily apparent to those skilled in the art.Typically, dosage is administered at lower levels and increased untilthe desired effect is achieved. When lipids are used to deliver theoligonucleotides, the amount of lipid compound that is administered canvary and generally depends upon the amount of oligonucleotide agentbeing administered. For example, the weight ratio of lipid compound tooligonucleotide agent is preferably from about 1:1 to about 15:1, with aweight ratio of about 5:1 to about 10:1 being more preferred. Generally,the amount of cationic lipid compound which is administered will varyfrom between about 0.1 milligram (mg) to about 1 gram (g). By way ofgeneral guidance, typically between about 0.1 mg and about 10 mg of theparticular oligonucleotide agent, and about 1 mg to about 100 mg of thelipid compositions, each per kilogram of patient body weight, isadministered, although higher and lower amounts can be used.

The agents of the invention are administered to subjects or contactedwith cells in a biologically compatible form suitable for pharmaceuticaladministration. By “biologically compatible form suitable foradministration” is meant that the oligonucleotide is administered in aform in which any toxic effects are outweighed by the therapeuticeffects of the oligonucleotide. In one embodiment, oligonucleotides canbe administered to subjects. Examples of subjects include mammals, e.g.,humans and other primates; cows, pigs, horses, and farming(agricultural) animals; dogs, cats, and other domesticated pets; mice,rats, and transgenic non-human animals.

Administration of an active amount of an oligonucleotide of the presentinvention is defined as an amount effective, at dosages and for periodsof time necessary to achieve the desired result. For example, an activeamount of an oligonucleotide may vary according to factors such as thetype of cell, the oligonucleotide used, and for in vivo uses the diseasestate, age, sex, and weight of the individual, and the ability of theoligonucleotide to elicit a desired response in the individual.Establishment of therapeutic levels of oligonucleotides within the cellis dependent upon the rates of uptake and efflux or degradation.Decreasing the degree of degradation prolongs the intracellularhalf-life of the oligonucleotide. Thus, chemically-modifiedoligonucleotides, e.g., with modification of the phosphate backbone, mayrequire different dosing.

The exact dosage of an oligonucleotide and number of doses administeredwill depend upon the data generated experimentally and in clinicaltrials. Several factors such as the desired effect, the deliveryvehicle, disease indication, and the route of administration, willaffect the dosage. Dosages can be readily determined by one of ordinaryskill in the art and formulated into the subject pharmaceuticalcompositions. Preferably, the duration of treatment will extend at leastthrough the course of the disease symptoms.

Dosage regim may be adjusted to provide the optimum therapeuticresponse. For example, the oligonucleotide may be repeatedlyadministered, e.g., several doses may be administered daily or the dosemay be proportionally reduced as indicated by the exigencies of thetherapeutic situation. One of ordinary skill in the art will readily beable to determine appropriate doses and schedules of administration ofthe subject oligonucleotides, whether the oligonucleotides are to beadministered to cells or to subjects.

Physical methods of introducing nucleic acids include injection of asolution containing the nucleic acid, bombardment by particles coveredby the nucleic acid, soaking the cell or organism in a solution of thenucleic acid, or electroporation of cell membranes in the presence ofthe nucleic acid. A viral construct packaged into a viral particle wouldaccomplish both efficient introduction of an expression construct intothe cell and transcription of nucleic acid encoded by the expressionconstruct. Other methods known in the art for introducing nucleic acidsto cells may be used, such as lipid-mediated carrier transport,chemical-mediated transport, such as calcium phosphate, and the like.Thus the nucleic acid may be introduced along with components thatperform one or more of the following activities: enhance nucleic aciduptake by the cell, inhibit annealing of single strands, stabilize thesingle strands, or other-wise increase inhibition of the target gene.

Nucleic acid may be directly introduced into the cell (i.e.,intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally or by inhalation, or may be introduced by bathing a cell ororganism in a solution containing the nucleic acid. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the nucleic acid may be introduced.

The cell with the target gene may be derived from or contained in anyorganism. The organism may a plant, animal, protozoan, bacterium, virus,or fungus. The plant may be a monocot, dicot or gymnosperm; the animalmay be a vertebrate or invertebrate. Preferred microbes are those usedin agriculture or by industry, and those that are pathogenic for plantsor animals.

Alternatively, vectors, e.g., transgenes encoding a siRNA of theinvention can be engineered into a host cell or transgenic animal usingart recognized techniques.

Another use for the nucleic acids of the present invention (or vectorsor transgenes encoding same) is a functional analysis to be carried outin eukaryotic cells, or eukaryotic non-human organisms, preferablymammalian cells or organisms and most preferably human cells, e.g. celllines such as HeLa or 293 or rodents, e.g. rats and mice. Byadministering a suitable nucleic acid of the invention which issufficiently complementary to a target mRNA sequence to directtarget-specific RNA interference, a specific knockout or knockdownphenotype can be obtained in a target cell, e.g. in cell culture or in atarget organism.

Thus, a further subject matter of the invention is a eukaryotic cell ora eukaryotic non-human organism exhibiting a target gene-specificknockout or knockdown phenotype comprising a fully or at least partiallydeficient expression of at least one endogenous target gene wherein saidcell or organism is transfected with at least one vector comprising DNAencoding an RNAi agent capable of inhibiting the expression of thetarget gene. It should be noted that the present invention allows atarget-specific knockout or knockdown of several different endogenousgenes due to the specificity of the RNAi agent.

Gene-specific knockout or knockdown phenotypes of cells or non-humanorganisms, particularly of human cells or non-human mammals may be usedin analytic to procedures, e.g. in the functional and/or phenotypicalanalysis of complex physiological processes such as analysis of geneexpression profiles and/or proteomes. Preferably the analysis is carriedout by high throughput methods using oligonucleotide based chips.

Assays of Oligonucleotide Stability

In some embodiments, the oligonucleotides of the invention arestabilized, i.e., substantially resistant to endonuclease andexonuclease degradation. An oligonucleotide is defined as beingsubstantially resistant to nucleases when it is at least about 3-foldmore resistant to attack by an endogenous cellular nuclease, and ishighly nuclease resistant when it is at least about 6-fold moreresistant than a corresponding oligonucleotide. This can be demonstratedby showing that the oligonucleotides of the invention are substantiallyresistant to nucleases using techniques which are known in the art.

One way in which substantial stability can be demonstrated is by showingthat the oligonucleotides of the invention function when delivered to acell, e.g., that they reduce transcription or translation of targetnucleic acid molecules, e.g., by measuring protein levels or bymeasuring cleavage of mRNA. Assays which measure the stability of targetRNA can be performed at about 24 hours post-transfection (e.g., usingNorthern blot techniques, RNase Protection Assays, or QC-PCR assays asknown in the art). Alternatively, levels of the target protein can bemeasured. Preferably, in addition to testing the RNA or protein levelsof interest, the RNA or protein levels of a control, non-targeted genewill be measured (e.g., actin, or preferably a control with sequencesimilarity to the target) as a specificity control. RNA or proteinmeasurements can be made using any art-recognized technique. Preferably,measurements will be made beginning at about 16-24 hours posttransfection. (M. Y. Chiang, et al. 1991. J Biol Chem. 266:18162-71; T.Fisher, et al. 1993. Nucleic Acids Research. 21 3857).

The ability of an oligonucleotide composition of the invention toinhibit protein synthesis can be measured using techniques which areknown in the art, for example, by detecting an inhibition in genetranscription or protein synthesis. For example, Nuclease Si mapping canbe performed. In another example, Northern blot analysis can be used tomeasure the presence of RNA encoding a particular protein. For example,total RNA can be prepared over a cesium chloride cushion (see, e.g.,Ausebel et al., 1987. Current Protocols in Molecular Biology (Greene &Wiley, New York)). Northern blots can then be made using the RNA andprobed (see, e.g., Id.). In another example, the level of the specificmRNA produced by the target protein can be measured, e.g., using PCR. Inyet another example, Western blots can be used to measure the amount oftarget protein present. In still another embodiment, a phenotypeinfluenced by the amount of the protein can be detected. Techniques forperforming Western blots are well known in the art, see, e.g., Chen etal. J. Biol. Chem. 271:28259.

In another example, the promoter sequence of a target gene can be linkedto a reporter gene and reporter gene transcription (e.g., as describedin more detail below) can be monitored. Alternatively, oligonucleotidecompositions that do not target a promoter can be identified by fusing aportion of the target nucleic acid molecule with a reporter gene so thatthe reporter gene is transcribed. By monitoring a change in theexpression of the reporter gene in the presence of the oligonucleotidecomposition, it is possible to determine the effectiveness of theoligonucleotide composition in inhibiting the expression of the reportergene. For example, in one embodiment, an effective oligonucleotidecomposition will reduce the expression of the reporter gene.

A “reporter gene” is a nucleic acid that expresses a detectable geneproduct, which may be RNA or protein. Detection of mRNA expression maybe accomplished by Northern blotting and detection of protein may beaccomplished by staining with antibodies specific to the protein.Preferred reporter genes produce a readily detectable product. Areporter gene may be operably linked with a regulatory DNA sequence suchthat detection of the reporter gene product provides a measure of thetranscriptional activity of the regulatory sequence. In preferredembodiments, the gene product of the reporter gene is detected by anintrinsic activity associated with that product. For instance, thereporter gene may encode a gene product that, by enzymatic activity,gives rise to a detectable signal based on color, fluorescence, orluminescence. Examples of reporter genes include, but are not limitedto, those coding for chloramphenicol acetyl transferase (CAT),luciferase, beta-galactosidase, and alkaline phosphatase.

One skilled in the art would readily recognize numerous reporter genessuitable for use in the present invention. These include, but are notlimited to, chloramphenicol acetyltransferase (CAT), luciferase, humangrowth hormone (hGH), and beta-galactosidase. Examples of such reportergenes can be found in F. A. Ausubel et al., Eds., Current Protocols inMolecular Biology, John Wiley & Sons, New York, (1989). Any gene thatencodes a detectable product, e.g., any product having detectableenzymatic activity or against which a specific antibody can be raised,can be used as a reporter gene in the present methods.

One reporter gene system is the firefly luciferase reporter system.(Gould, S. J., and Subramani, S. 1988. Anal. Biochem., 7:404-408incorporated herein by reference). The luciferase assay is fast andsensitive. In this assay, a lysate of the test cell is prepared andcombined with ATP and the substrate luciferin. The encoded enzymeluciferase catalyzes a rapid, ATP dependent oxidation of the substrateto generate a light-emitting product. The total light output is measuredand is proportional to the amount of luciferase present over a widerange of enzyme concentrations.

CAT is another frequently used reporter gene system; a major advantageof this system is that it has been an extensively validated and iswidely accepted as a measure of promoter activity. (Gorman C. M.,Moffat, L. F., and Howard, B. H.1982. Mol. Cell. Biol., 2:1044-1051). Inthis system, test cells are transfected with CAT expression vectors andincubated with the candidate substance within 2-3 days of the initialtransfection. Thereafter, cell extracts are prepared. The extracts areincubated with acetyl CoA and radioactive chloramphenicol. Following theincubation, acetylated chloramphenicol is separated from nonacetylatedform by thin layer chromatography. In this assay, the degree ofacetylation reflects the CAT gene activity with the particular promoter.

Another suitable reporter gene system is based on immunologic detectionof hGH. This system is also quick and easy to use. (Selden, R.,Burke-Howie, K. Rowe, M. E., Goodman, H. M., and Moore, D. D. (1986),Mol. Cell, Biol., 6:3173-3179 incorporated herein by reference). The hGHsystem is advantageous in that the expressed hGH polypeptide is assayedin the media, rather than in a cell extract. Thus, this system does notrequire the destruction of the test cells. It will be appreciated thatthe principle of this reporter gene system is not limited to hGH butrather adapted for use with any polypeptide for which an antibody ofacceptable specificity is available or can be prepared.

In one embodiment, nuclease stability of a double-strandedoligonucleotide of the invention is measured and compared to a control,e.g., an RNAi molecule typically used in the art (e.g., a duplexoligonucleotide of less than 25 nucleotides in length and comprising 2nucleotide base overhangs) or an unmodified RNA duplex with blunt ends.

The target RNA cleavage reaction achieved using the siRNAs of theinvention is highly sequence specific. Sequence identity may determinedby sequence comparison and alignment algorithms known in the art. Todetermine the percent identity of two nucleic acid sequences (or of twoamino acid sequences), the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the first sequence or secondsequence for optimal alignment). A preferred, non-limiting example of alocal alignment algorithm utilized for the comparison of sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. Additionally, numerous commercial entities, such asDharmacon, and Invitrogen provide access to algorithms on their website.The Whitehead Institute also offers a free siRNA Selection Program.Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or even 100% sequence identity, between the siRNA and theportion of the target gene is preferred. Alternatively, the siRNA may bedefined functionally as a nucleotide sequence (or oligonucleotidesequence) that is capable of hybridizing with a portion of the targetgene transcript. Examples of stringency conditions for polynucleotidehybridization are provided in Sambrook, J., E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11,and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al.,eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporatedherein by reference.

Therapeutic Use

By inhibiting the expression of a gene, the oligonucleotide compositionsof the present invention can be used to treat any disease involving theexpression of a protein. Examples of diseases that can be treated byoligonucleotide compositions, just to illustrate, include: cancer,retinopathies, autoimmune diseases, inflammatory diseases (i.e., ICAM-1related disorders, Psoriasis, Ulcerative Colitus, Crohn's disease),viral diseases (i.e., HIV, Hepatitis C), miRNA disorders, andcardiovascular diseases.

In one embodiment, in vitro treatment of cells with oligonucleotides canbe used for ex vivo therapy of cells removed from a subject (e.g., fortreatment of leukemia or viral infection) or for treatment of cellswhich did not originate in the subject, but are to be administered tothe subject (e.g., to eliminate transplantation antigen expression oncells to be transplanted into a subject). In addition, in vitrotreatment of cells can be used in non-therapeutic settings, e.g., toevaluate gene function, to study gene regulation and protein synthesisor to evaluate improvements made to oligonucleotides designed tomodulate gene expression or protein synthesis. In vivo treatment ofcells can be useful in certain clinical settings where it is desirableto inhibit the expression of a protein. There are numerous medicalconditions for which antisense therapy is reported to be suitable (see,e.g., U.S. Pat. No. 5,830,653) as well as respiratory syncytial virusinfection (WO 95/22,553) influenza virus (WO 94/23,028), andmalignancies (WO 94/08,003). Other examples of clinical uses ofantisense sequences are reviewed, e.g., in Glaser. 1996. GeneticEngineering News 16:1. Exemplary targets for cleavage byoligonucleotides include, e.g., protein kinase Ca, ICAM-1, c-raf kinase,p53, c-myb, and the bcr/abl fusion gene found in chronic myelogenousleukemia.

The subject nucleic acids can be used in RNAi-based therapy in anyanimal having RNAi pathway, such as human, non-human primate, non-humanmammal, non-human vertebrates, rodents (mice, rats, hamsters, rabbits,etc.), domestic livestock animals, pets (cats, dogs, etc.), Xenopus,fish, insects (Drosophila, etc.), and worms (C. elegans), etc.

The invention provides methods for inhibiting or preventing in asubject, a disease or condition associated with an aberrant or unwantedtarget gene expression or activity, by administering to the subject anucleic acid of the invention. If appropriate, subjects are firsttreated with a priming agent so as to be more responsive to thesubsequent RNAi therapy. Subjects at risk for a disease which is causedor contributed to by aberrant or unwanted target gene expression oractivity can be identified by, for example, any or a combination ofdiagnostic or prognostic assays known in the art. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the target gene aberrancy, such that a disease ordisorder is prevented or, alternatively, delayed in its progression.Depending on the type of target gene aberrancy, for example, a targetgene, target gene agonist or target gene antagonist agent can be usedfor treating the subject.

In another aspect, the invention pertains to methods of modulatingtarget gene expression, protein expression or activity for therapeuticpurposes. Accordingly, in an exemplary embodiment, the methods of theinvention involve contacting a cell capable of expressing target genewith a nucleic acid of the invention that is specific for the targetgene or protein (e.g., is specific for the mRNA encoded by said gene orspecifying the amino acid sequence of said protein) such that expressionor one or more of the activities of target protein is modulated. Thesemethods can be performed in vitro (e.g., by culturing the cell with theagent), in vivo (e.g., by administering the agent to a subject), or exvivo. The subjects may be first treated with a priming agent so as to bemore responsive to the subsequent RNAi therapy if desired. As such, thepresent invention provides methods of treating a subject afflicted witha disease or disorder characterized by aberrant or unwanted expressionor activity of a target gene polypeptide or nucleic acid molecule.Inhibition of target gene activity is desirable in situations in whichtarget gene is abnormally unregulated and/or in which decreased targetgene activity is likely to have a beneficial effect.

Thus the therapeutic agents of the invention can be administered tosubjects to treat (prophylactically or therapeutically) disordersassociated with aberrant or unwanted target gene activity. Inconjunction with such treatment, pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) may be considered. Differencesin metabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a therapeutic agent as wellas tailoring the dosage and/or therapeutic regimen of treatment with atherapeutic agent. Pharmacogenomics deals with clinically significanthereditary variations in the response to drugs due to altered drugdisposition and abnormal action in affected persons.

For the purposes of the invention, ranges may be expressed herein asfrom “about” one particular value, and/or to “about” another particularvalue. When such a range is expressed, another embodiment includes fromthe one particular value and/or to the other particular value.Similarly, when values are expressed as approximations, by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment. It will be further understood that theendpoints of each of the ranges are significant both in relation to theother endpoint, and independently of the other endpoint.

Moreover, for the purposes of the present invention, the term “a” or“an” entity refers to one or more of that entity; for example, “aprotein” or “a nucleic acid molecule” refers to one or more of thosecompounds or at least one compound. As such, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably. Furthermore, a compound “selectedfrom the group consisting of” refers to one or more of the compounds inthe list that follows, including mixtures (i.e., combinations) of two ormore of the compounds. According to the present invention, an isolated,or biologically pure, protein or nucleic acid molecule is a compoundthat has been removed from its natural milieu. As such, “isolated” and“biologically pure” do not necessarily reflect the extent to which thecompound has been purified. An isolated compound of the presentinvention can be obtained from its natural source, can be produced usingmolecular biology techniques or can be produced by chemical synthesis.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1 Inhibition of Gene Expression using Minimum LengthTrigger RNAs

Transfection of Minimum Length Trigger (mlt) RNA

mltRNA constructs were chemically synthesized (Integrated DNATechnologies, Coralville, Iowa) and transfected into HEK293 cells (ATCC,Manassas, Va.) using the Lipofectamine RNAiMAX (Invitrogen, Carlsbad,Calif.) reagent according to manufacturer's instructions. In brief, RNAwas diluted to a 12× concentration and then combined with a 12×concentration of Lipofectamine RNAiMAX to complex. The RNA andtransfection reagent were allowed to complex at room temperature for 20minutes and make a 6× concentration. While complexing, HEK293 cells werewashed, trypsinized and counted. The cells were diluted to aconcentration recommended by the manufacturer and previously describedconditions which was at 1×10⁵ cells/ml. When RNA had completedcomplexing with the RNAiMAX transfection reagent, 20 ul of the complexeswere added to the appropriate well of the 96-well plate in triplicate.Cells were added to each well (100 ul volume) to make the final cellcount per well at 1×10⁴ cells/well. The volume of cells diluted the 6×concentration of complex to 1× which was equal to a concentration noted(between 10-0.05 nM). Cells were incubated for 24 or 48 hours undernormal growth conditions.

After 24 or 48 hour incubation cells were lysed and gene silencingactivity was measured using the QuantiGene assay (Panomics, Freemont,Calif.) which employs bDNA hybridization technology. The assay wascarried out according to manufacturer's instructions.

ΔG Calculation

ΔG was calculated using Mfold, available through the Mfold internet site(http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi). Methods forcalculating ΔG are described in, and are incorporated by reference from,the following references: Zuker, M. (2003) Nucleic Acids Res.,31(13):3406-15; Mathews, D. H., Sabina, J., Zuker, M. and Turner, D. H.(1999) J. Mol. Biol. 288:911-940; Mathews, D. H., Disney, M. D., Childs,J. L., Schroeder, S. J., Zuker, M., and Turner, D. H. (2004) Proc. Natl.Acad. Sci. 101:7287-7292; Duan, S., Mathews, D. H., and Turner, D. H.(2006) Biochemistry 45:9819-9832; Wuchty, S., Fontana, W., Hofacker, I.L., and Schuster, P. (1999) Biopolymers 49:145-165.

Example 2 Optimization of sd-rxRNA^(nano) Molecules for Gene Silencing

Asymmetric double stranded RNAi molecules, with minimal double strandedregions, were developed herein and are highly effective at genesilencing. These molecules can contain a variety of chemicalmodifications on the sense and/or anti-sense strands, and can beconjugated to sterol-like compounds such as cholesterol.

FIGS. 1-3 present schematics of RNAi molecules associated with theinvention. In the asymmetric molecules, which contain a sense andanti-sense strand, either of the strands can be the longer strand.Either strand can also contain a single-stranded region. There can alsobe mismatches between the sense and anti-sense strand, as indicated inFIG. 1D. Preferably, one end of the double-stranded molecule is eitherblunt-ended or contains a short overhang such as an overhang of onenucleotide. FIG. 2 indicates types of chemical modifications applied tothe sense and anti-sense strands including 2′F, 2′OMe, hydrophobicmodifications and phosphorothioate modifications. Preferably, the singlestranded region of the molecule contains multiple phosphorothioatemodifications. Hydrophobicity of molecules can be increased using suchcompounds as 4-pyridyl at 5-U, 2-pyridyl at 5-U, isobutyl at 5-U andindolyl at 5-U (FIG. 2). Proteins or peptides such as protamine (orother Arg rich peptides), spermidine or other similar chemicalstructures can also be used to block duplex charge and facilitatecellular entry (FIG. 3). Increased hydrophobicity can be achievedthrough either covalent or non-covalent modifications. Severalpositively charged chemicals, which might be used for polynucleotidecharge blockage are depicted in FIG. 4.

Chemical modifications of polynucleotides, such as the guide strand in aduplex molecule, can facilitate RISC entry. FIG. 5 depicts singlestranded polynucleotides, representing a guide strand in a duplexmolecule, with a variety of chemical modifications including 2′ d,2′OMe, 2′F, hydrophobic modifications, phosphorothioate modifications,and attachment of conjugates such as “X” in FIG. 5, where X can be asmall molecule with high affinity to a PAZ domain, or sterol-typeentity. Similarly, FIG. 6 depicts single stranded polynucleotides,representing a passenger strand in a duplex molecule, with proposedstructural and chemical compositions of RISC substrate inhibitors.Combinations of chemical modifications can ensure efficient uptake andefficient binding to preloaded RISC complexes.

FIG. 7 depicts structures of polynucleotides with sterol-type moleculesattached, where R represents a polycarbonic tail of 9 carbons or longer.FIG. 8 presents examples of naturally occurring phytosterols with apolycarbon chain longer than 8 attached at position 17. More than 250different types of phytosterols are known. FIG. 9 presents examples ofsterol-like structures with variations in the sizes of the polycarbonchains attached at position 17. FIG. 91 presents further examples ofsterol-type molecules that can be used as a hydrophobic entity in placeof cholesterol. FIG. 92 presents further examples of hydrophobicmolecules that might be used as hydrophobic entities in place ofcholestesterol. Optimization of such characteristics can improve uptakeproperties of the RNAi molecules. FIG. 10 presents data adapted fromMartins et al. (J Lipid Research), showing that the percentage of liveruptake and plasma clearance of lipid emulsions containing sterol-typemolecules is directly affected by the size of the attached polycarbonchain at position 17. FIG. 11 depicts a micelle formed from a mixture ofpolynucleotides attached to hydrophobic conjugates and fatty acids. FIG.12 describes how alteration in lipid composition can affectpharmacokinetic behavior and tissue distribution of hydrophobicallymodified and/or hydrophobically conjugated polynucleotides. Inparticular, the use of lipid mixtures that are enriched in linoleic acidand cardiolipin results in preferential uptake by cardiomyocites.

FIG. 13 depicts examples of RNAi constructs and controls designed totarget MAP4K4 expression. FIGS. 14 and 15 reveal that RNAi constructswith minimal duplex regions (such as duplex regions of approximately 13nucleotides) are effective in mediating RNA silencing in cell culture.Parameters associated with these RNA molecules are shown in FIG. 16.FIG. 17 depicts examples of RNAi constructs and controls designed totarget SOD1 expression. FIGS. 18 and 19 reveal the results of genesilencing experiments using these RNAi molecules to target SOD1 incells. FIG. 20 presents a schematic indicating that RNA molecules withdouble stranded regions that are less than 10 nucleotides are notcleaved by Dicer, and FIG. 21 presents a schematic of a hypotheticalRNAi model for RNA induced gene silencing.

The RNA molecules described herein were subject to a variety of chemicalmodifications on the sense and antisense strands, and the effects ofsuch modifications were observed. RNAi molecules were synthesized andoptimized through testing of a variety of modifications. In firstgeneration optimization, the sense (passenger) and anti-sense (guide)strands of the sd-rxRNA'° molecules were modified for example throughincorporation of C and U 2′OMe modifications, 2′F modifications,phosphorothioate modifications, phosphorylation, and conjugation ofcholesterol. Molecules were tested for inhibition of MAP4K4 expressionin cells including HeLa, primary mouse hepatocytes and primary humanhepatocytes through both lipid-mediated and passive uptake transfection.

FIG. 22 reveals that chemical modifications can enhance gene silencing.In particular, modifying the guide strand with 2′F UC modifications, andwith a stretch of phosphorothioate modifications, combined with completeCU O′Me modification of the passenger strands, resulted in moleculesthat were highly effective in gene silencing. The effect of chemicalmodification on in vitro efficacy in un-assisted delivery in HeLa cellswas also examined. FIG. 23 reveals that compounds lacking any of 2′F,2′OMe, a stretch of phosphorothioate modifications, or cholesterolconjugates, were completely inactive in passive uptake. A combination ofall 4 types of chemical modifications, for example in compound 12386,was found to be highly effective in gene silencing. FIG. 24 also showsthe effectiveness of compound 12386 in gene silencing.

Optimization of the length of the oligonucleotide was also investigated.FIGS. 25 and 26 reveal that oligonucleotides with a length of 21nucleotides were more effective than oligonucleotides with a length of25 nucleotides, indicating that reduction in the size of an RNA moleculecan improve efficiency, potentially by assisting in its uptake.Screening was also conducted to optimize the size of the duplex regionof double stranded RNA molecules. FIG. 88 reveals that compounds withduplexes of 10 nucleotides were effective in inducing gene silencing.Positioning of the sense strand relative to the guide strand can also becritical for silencing gene expression (FIG. 89). In this assay, a bluntend was found to be most effective. 3′ overhangs were tolerated, but 5′overhangs resulted in a complete loss of functionality. The guide strandcan be effective in gene silencing when hybridized to a sense strand ofvarying lengths (FIG. 90). In this assay presented in FIG. 90, thecompounds were introduced into HeLa cells via lipid mediatedtransfection.

The importance of phosphorothioate content of the RNA molecule forunassisted delivery was also investigated. FIG. 27 presents the resultsof a systematic screen that identified that the presence of at least2-12 phosphorothioates in the guide strand as being highly advantageousfor achieving uptake, with 4-8 being the preferred number. FIG. 27 alsoshows that presence or absence of phosphorothioate modifications in thesense strand did not alter efficacy.

FIGS. 28-29 reveal the effects of passive uptake of RNA compounds ongene silencing in primary mouse hepatocytes. nanoRNA molecules werefound to be highly effective, especially at a concentration of 1 μM(FIG. 28). FIGS. 30 and 31 reveal that the RNA compounds associated withthe invention were also effective in gene silencing following passiveuptake in primary human hepatocytes. The cellular localization of theRNA molecules associated with the invention was examined and compared tothe localization of Chol-siRNA (Alnylam) molecules, as shown in FIGS. 32and 33.

A summary of 1^(st) generation sd-rxRNA molecules is presented in FIG.21. Chemical modifications were introduced into the RNA molecules, atleast in part, to increase potency, such as through optimization ofnucleotide length and phosphorothioate content, to reduce toxicity, suchas through replacing 2′F modifications on the guide strand with othermodifications, to improve delivery such as by adding or conjugating theRNA molecules to linker and sterol modalities, and to improve the easeof manufacturing the RNA molecules. FIG. 35 presents schematicdepictions of some of the chemical modifications that were screened in1^(st) generation molecules. Parameters that were optimized for theguide strand included nucleotide length (e.g., 19, 21 and 25nucleotides), phosphorothioate content (e.g., 0-18 phosphorothioatelinkages) and replacement of 2′F groups with 2′OMe and 5 Me C orriboThymidine. Parameters that were optimized for the sense strandincluded nucleotide length (e.g., 11, 13 and 19 nucleotides),phosphorothioate content (e.g., 0-4 phosphorothioate linkages), and2′OMe modifications. FIG. 36 summarizes parameters that were screened.For example, the nucleotide length and the phosphorothioate tail lengthwere modified and screened for optimization, as were the additions of2′OMe C and U modifications. Guide strand length and the length of thephosphorothioate modified stretch of nucleotides were found to influenceefficacy (FIGS. 37-38). Phosphorothioate modifications were tolerated inthe guide strand and were found to influence passive uptake (FIGS.39-42).

FIG. 43 presents a schematic revealing guide strand chemicalmodifications that were screened. FIGS. 44 and 45 reveal that 2′ OMemodifications were tolerated in the 3′ end of the guide strand. Inparticular, 2′OMe modifications in positions 1 and 11-18 were welltolerated. The 2′OMe modifications in the seed area were tolerated butresulted in slight reduction of efficacy. Ribo-modifications in the seedwere also well tolerated. These data indicate that the moleculesassociated with the invention offer the significant advantage of havingreduced or no 2′F modification content. This is advantageous because 2′Fmodifications are thought to generate toxicity in vivo. In someinstances, a complete substitution of 2′F modifications with 2′OMe wasfound to lead to some reduction in potency. However, the 2′ OMesubstituted molecules were still very active. A molecule with 50%reduction in 2′F content (including at positions 11, 16-18 which werechanged to 2′OMe modifications), was found to have comparable efficacyto a compound with complete 2′F C and U modification. 2′OMe modificationin position was found in some instances to reduce efficacy, althoughthis can be at least partially compensated by 2′OMe modification inposition 1 (with chemical phosphate). In some instances, 5 Me C and/orribothymidine substitution for 2′F modifications led to a reduction inpassive uptake efficacy, but increased potency in lipid mediatedtransfections compared to 2′F modifications. Optimization results forlipid mediated transfection were not necessarily the same as for passiveuptake.

Modifications to the sense strand were also developed and tested, asdepicted in FIG. 46. FIG. 47 reveals that in some instances, a sensestrand length between 10-15 bases was found to be optimal. For themolecules tested in FIG. 47, an increase in the sense strand lengthresulted in reduction of passive uptake, however an increase in sensestrand length may be tolerated for some compounds. FIG. 47 also revealsthat LNA modification of the sense strand demonstrated similar efficacyto non-LNA containing compounds. In general, the addition of LNA orother thermodynamically stabilizing compounds has been found to bebeneficial, in some instances resulting in converting non-functionalsequences to functional sequences. FIG. 48 also presents data on sensestrand length optimization, while FIG. 49 shows that phosphorothioatemodification of the sense strand is not required for passive uptake.

Based on the above-described optimization experiments, 2^(nd) generationRNA molecules were developed. As shown in FIG. 50, these moleculescontained reduced phosphorothioate modification content and reduced 2′Fmodification content, relative to 1^(st) generation RNA molecules.Significantly, these RNA molecules exhibit spontaneous cellular uptakeand efficacy without a delivery vehicle (FIG. 51). These molecules canachieve self-delivery (i.e., with no transfection reagent) and followingself-delivery can exhibit nanomolar activity in cell culture. Thesemolecules can also be delivered using lipid-mediated transfection, andexhibit picomolar activity levels following transfection. Significantly,these molecules exhibit highly efficient uptake, 95% by most cells incell culture, and are stable for more than three days in the presence of100% human serum. These molecules are also highly specific and exhibitlittle or no immune induction. FIGS. 52 and 53 reveal the significanceof chemical modifications and the configurations of such modificationsin influencing the properties of the RNA molecules associated with theinvention.

Linker chemistry was also tested in conjunction with the RNA moleculesassociated with the invention. As depicted in FIG. 54, 2^(nd) generationRNA molecules were synthesized with sterol-type molecules attachedthrough TEG and amino caproic acid linkers. Both linkers showedidentical potency. This functionality of the RNA molecules, independentof linker chemistry offers additional advantages in terms of scale upand synthesis and demonstrates that the mechanism of function of theseRNA molecules is very different from other previously described RNAmolecules.

Stability of the chemically modified sd-rxRNA molecules described hereinin human serum is shown in FIG. 55 in comparison to unmodified RNA. Theduplex molecules were incubated in 75% serum at 37° C. for the indicatedperiods of time. The level of degradation was determined by running thesamples on non-denaturing gels and staining with SYBGR.

FIGS. 56 and 57 present data on cellular uptake of the sd-rxRNAmolecules. FIG. 56 shows that minimizing the length of the RNA moleculeis importance for cellular uptake, while FIG. 57 presents data showingtarget gene silencing after spontaneous cellular uptake in mousePEC-derived macrophages. FIG. 58 demonstrates spontaneous uptake andtarget gene silencing in primary cells. FIG. 59 shows the results ofdelivery of sd-rxRNA molecules associated with the invention to RPEcells with no formulation. Imaging with Hoechst and DY547 reveals theclear presence of a signal representing the RNA molecule in the sd-rxRNAsample, while no signal is detectable in the other samples including thesamples competing a competing conjugate, an rxRNA, and an untransfectedcontrol. FIG. 60 reveals silencing of target gene expression in RPEcells treated with sd-rxRNA molecules associated with the inventionfollowing 24-48 hours without any transfection formulation.

FIG. 61 shows further optimization of the chemical/structuralcomposition of sd-rxRNA compounds. In some instances, preferredproperties included an antisense strand that was 17-21 nucleotides long,a sense strand that was 10-15 nucleotides long, phosphorothioatemodification of 2-12 nucleotides within the single stranded region ofthe molecule, preferentially phosphorothioate modification of 6-8nucleotides within the single stranded region, and 2′OMe modification atthe majority of positions within the sense strand, with or withoutphosphorothioate modification. Any linker chemistry can be used toattach the hydrophobic moiety, such as cholesterol, to the 3′ end of thesense strand. Version Glib molecules, as shown in FIG. 61, have no 2′Fmodifications. Significantly, there is was no impact on efficacy inthese molecules.

FIG. 62 demonstrates the superior performance of sd-rxRNA compoundscompared to compounds published by Wolfrum et. al. Nature Biotech, 2007.Both generation I and II compounds (GI and GIIa) developed herein showgreat efficacy in reducing target gene expression. By contrast, when thechemistry described in Wolfrum et al. (all oligos contain cholesterolconjugated to the 3′ end of the sense strand) was applied to the samesequence in a context of conventional siRNA (19 bp duplex with twooverhang) the compound was practically inactive. These data emphasizethe significance of the combination of chemical modifications andassymetrical molecules described herein, producing highly effective RNAcompounds.

FIG. 63 shows localization of sd-rxRNA molecules developed hereincompared to localization of other RNA molecules such as those describedin Soutschek et al. (2004) Nature, 432:173. sd-rxRNA moleculesaccumulate inside the cells whereas competing conjugate RNAs accumulateon the surface of cells. Significantly, FIG. 64 shows that sd-rxRNAmolecules, but not competitor molecules such as those described inSoutschek et al. are internalized within minutes. FIG. 65 compareslocalization of sd-rxRNA molecules compared to regularsiRNA-cholesterol, as described in Soutschek et al. A signalrepresenting the RNA molecule is clearly detected for the sd-rxRNAmolecule in tissue culture RPE cells, following local delivery tocompromised skin, and following systemic delivery where uptake to theliver is seen. In each case, no signal is detected for the regularsiRNA-cholesterol molecule. The sd-rxRNA molecule thus has drasticallybetter cellular and tissue uptake characteristics when compared toconventional cholesterol conjugated siRNAs such as those described inSoutschek et al. The level of uptake is at least order of magnitudehigher and is due at least in part to the unique combination ofchemistries and conjugated structure. Superior delivery of sd-rxRNArelative to previously described RNA molecules is also demonstrated inFIGS. 66 and 67.

Based on the analysis of 2^(nd) generation RNA molecules associated withthe invention, a screen was performed to identify functional moleculesfor targeting the SPP1/PPIB gene. As revealed in FIG. 68, severaleffective molecules were identified, with 14131 being the mosteffective. The compounds were added to A-549 cells and then the level ofSPP1/PPIB ratio was determined by B-DNA after 48 hours.

FIG. 69 reveals efficient cellular uptake of sd-rxRNA within minutes ofexposure. This is a unique characteristics of these molecules, notobserved with any other RNAi compounds. Compounds described in Soutscheket al. were used as negative controls. FIG. 70 reveals that the uptakeand gene silencing of the sd-rxRNA is effective in multiple differentcell types including SH-SY5Y neuroblastoma derived cells, ARPE-19(retinal pigment epithelium) cells, primary hepatocytes, and primarymacrophages. In each case silencing was confirmed by looking at targetgene expression by a Branched DNA assay.

FIG. 70 reveals that sd-rxRNA is active in the presence or absence ofserum. While a slight reduction in efficacy (2-5 fold) was observed inthe presence of serum, this small reduction in efficacy in the presenceof serum differentiate the sd-rxRNA molecules from previously describedmolecules which exhibited a larger reduction in efficacy in the presenceof serum. This demonstrated level of efficacy in the presence of serumcreates a foundation for in vivo efficacy.

FIG. 72 reveals efficient tissue penetration and cellular uptake uponsingle intradermal injection. This data indicates the potential of thesd-rxRNA compounds described herein for silencing genes in anydermatology applications, and also represents a model for local deliveryof sd-rxRNA compounds. FIG. 73 also demonstrates efficient cellularuptake and in vivo silencing with sd-rxRNA following intradermalinjection. Silencing is determined as the level of MAP4K4 knockdown inseveral individual biopsies taken from the site of injection as comparedto biopsies taken from a site injected with a negative control. FIG. 74reveals that sd-rxRNA compounds has improved blood clearance and inducedeffective gene silencing in vivo in the liver upon systemicadministration. In comparison to the RNA molecules described bySoutschek et al., the level of liver uptake at identical dose level isat least 50 fold higher with the sd-rxRNA molecules. The uptake resultsin productive silencing. sd-rxRNA compounds are also characterized byimproved blood clearance kinetics.

The effect of 5-Methyl C modifications was also examined. FIG. 75demonstrates that the presence of 5-Methyl C in an RNAi moleculeresulted in increased potency in lipid mediated transfection. Thissuggests that hydrophobic modification of Cs and Us in an RNAi moleculecan be beneficial. These types of modifications can also be used in thecontext 2′ ribose modified bases to ensure optimal stability andefficacy. FIG. 76 presents data showing that incorporation of 5-Methyl Cand/or ribothymidine in the guide strand can in some instances reduceefficacy.

FIG. 77 reveals that sd-rxRNA molecules are more effective thancompetitor molecules such as molecules described in Soutschek et al., insystemic delivery to the liver. A signal representing the RNA moleculeis clearly visible in the sample containing sd-rxRNA, while no signalrepresenting the RNA molecule is visible in the sample containing thecompetitor RNA molecule.

The addition of hydrophobic conjugates to the sd-rxRNA molecules wasalso explored (FIGS. 78-83). FIG. 78 presents schematics demonstrating5-uridyl modifications with improved hydrophobicity characteristics.Incorporation of such modifications into sd-rxRNA compounds can increasecellular and tissue uptake properties. FIG. 78B presents a new type ofRNAi compound modification which can be applied to compounds to improvecellular uptake and pharmacokinetic behavior. Significantly, this typeof modification, when applied to sd-rxRNA compounds, may contribute tomaking such compounds orally available. FIG. 79 presents schematicsrevealing the structures of synthesized modified sterol-type molecules,where the length and structure of the C17 attached tail is modified.Without wishing to be bound by any theory, the length of the C17attached tail may contribute to improving in vitro and in vivo efficacyof sd-rxRNA compounds.

FIG. 80 presents a schematic demonstrating the lithocholic acid route tolong side chain cholesterols. FIG. 81 presents a schematic demonstratinga route to 5-uridyl phosphoramidite synthesis. FIG. 82 presents aschematic demonstrating synthesis of tri-functional hydroxyprolinollinker for 3′-cholesterol attachment. FIG. 83 presents a schematicdemonstrating synthesis of solid support for the manufacture of ashorter asymmetric RNAi compound strand.

A screen was conducted to identify compounds that could effectivelysilence expression of SPP1 (Osteopontin). Compounds targeting SPP1 wereadded to A549 cells (using passive transfection), and the level of SPP1expression was evaluated at 48 hours. Several novel compounds effectivein SPP1 silencing were identified. Compounds that were effective insilencing of SPP1 included 14116, 14121, 14131, 14134, 14139, 14149, and14152 (FIGS. 84-86). The most potent compound in this assay was 14131(FIG. 84). The efficacy of these sd-rxRNA compounds in silencing SPP1expression was independently validated (FIG. 85).

A similar screen was conducted to identify compounds that couldeffectively silence expression of CTGF (FIGS. 86-87). Compounds thatwere effective in silencing of CTGF included 14017, 14013, 14016, 14022,14025, 14027.

Methods

Transfection of sd-rxRNA^(nano)

Lipid Mediated Transfection

sd-rxRNA^(nano) constructs were chemically synthesized (Dharmacon,Lafayette, Colo.) and transfected into HEK293 cells (ATCC, Manassas,Va.) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, Calif.)according to the manufacturer's instructions. In brief, RNA was dilutedto a 12× concentration in Opti-MEM®1 Reduced Serum Media (Invitrogen,Carlsbad, Calif.) and then combined with a 12× concentration ofLipofectamine RNAiMAX. The RNA and transfection reagent were allowed tocomplex at room temperature for 20 minutes and make a 6× concentration.While complexing, HEK293 cells were washed, trypsinized and counted. Thecells were diluted to a concentration recommended by the manufacturerand previously described of 1×10⁵ cells/ml. When RNA had completedcomplexing with the RNAiMAX transfection reagent, 20 ul of the complexeswere added to the appropriate well of the 96-well plate in triplicate.Cells were added to each well (100 ul volume) to make the final cellcount per well 1×10⁴ cells/well. The volume of cells diluted the 6×concentration of complex to 1× (between 10-0.05 nM). Cells wereincubated for 24 or 48 hours under normal growth conditions. After 24 or48 hour incubation, cells were lysed and gene silencing activity wasmeasured using the QuantiGene assay (Panomics, Freemont, Calif.) whichemploys bDNA hybridization technology. The assay was carried outaccording to manufacturer's instructions.

Passive Uptake Transfection

sd-rxRNA^(nano) constructs were chemically synthesized (Dharmacon,Lafayette, Colo.). 24 hours prior to transfection, HeLa cells (ATCC,Manassas, Va.) were plated at 1×10⁴ cells/well in a 96 well plate undernormal growth conditions (DMEM, 10% FBS and 1% Penicillin andStreptomycin). Prior to transfection of HeLa cells, sd-rxRNA^(nano) werediluted to a final concentration of 0.01 uM to 1 uM in Accell siRNADelivery Media (Dharmacon, Lafayette, Colo.). Normal growth media wasaspirated off cells and 100 uL of Accell Delivery media containing theappropriate concentration of sd-rxRNAnano was applied to the cells. 48hours post transfection, delivery media was aspirated off the cells andnormal growth media was applied to cells for an additional 24 hours.

After 48 or 72 hour incubation, cells were lysed and gene silencingactivity was measured using the QuantiGene assay (Panomics, Freemont,Calif.) according to manufacturer's instructions.

TABLE 1 Oligo Accession Gene ID Number Number number Gene Name SymbolAPOB-10167- 12138 NM_000384 Apolipoprotein B (including Ag(x) APOB20-12138 antigen) APOB-10167- 12139 NM_000384 Apolipoprotein B(including Ag(x) APOB 20-12139 antigen) MAP4K4- 12266 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-13- Kinase Kinase Kinase 4(MAP4K4), 12266 transcript variant 1 MAP4K4- 12293 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-16- Kinase Kinase Kinase 4(MAP4K4), 12293 transcript variant 1 MAP4K4- 12383 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-16- Kinase Kinase Kinase 4(MAP4K4), 12383 transcript variant 1 MAP4K4- 12384 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-16- Kinase Kinase Kinase 4(MAP4K4), 12384 transcript variant 1 MAP4K4- 12385 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-16- Kinase Kinase Kinase 4(MAP4K4), 12385 transcript variant 1 MAP4K4- 12386 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-16- Kinase Kinase Kinase 4(MAP4K4), 12386 transcript variant 1 MAP4K4- 12387 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-16- Kinase Kinase Kinase 4(MAP4K4), 12387 transcript variant 1 MAP4K4- 12388 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-15- Kinase Kinase Kinase 4(MAP4K4), 12388 transcript variant 1 MAP4K4- 12432 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-13- Kinase Kinase Kinase 4(MAP4K4), 12432 transcript variant 1 MAP4K4- 12266.2 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-13- Kinase Kinase Kinase 4(MAP4K4), 12266.2 transcript variant 1 APOB--21- 12434 NM_000384Apolipoprotein B (including Ag(x) APOB 12434 antigen) APOB--21- 12435NM_000384 Apolipoprotein B (including Ag(x) APOB 12435 antigen) MAP4K4-12451 NM_004834 Mitogen-Activated Protein Kinase MAP4K4 2931-16- KinaseKinase Kinase 4 (MAP4K4), 12451 transcript variant 1 MAP4K4- 12452NM_004834 Mitogen-Activated Protein Kinase MAP4K4 2931-16- Kinase KinaseKinase 4 (MAP4K4), 12452 transcript variant 1 MAP4K4- 12453 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-16- Kinase Kinase Kinase 4(MAP4K4), 12453 transcript variant 1 MAP4K4- 12454 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-17- Kinase Kinase Kinase 4(MAP4K4), 12454 transcript variant 1 MAP4K4- 12455 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-17- Kinase Kinase Kinase 4(MAP4K4), 12455 transcript variant 1 MAP4K4- 12456 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-19- Kinase Kinase Kinase 4(MAP4K4), 12456 transcript variant 1 --27-12480 12480 --27-12481 12481APOB-10167- 12505 NM_000384 Apolipoprotein B (including Ag(x) APOB21-12505 antigen) APOB-10167- 12506 NM_000384 Apolipoprotein B(including Ag(x) APOB 21-12506 antigen) MAP4K4- 12539 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-16- Kinase Kinase Kinase 4(MAP4K4), 12539 transcript variant 1 APOB-10167- 12505.2 NM_000384Apolipoprotein B (including Ag(x) APOB 21-12505.2 antigen) APOB-10167-12506.2 NM_000384 Apolipoprotein B (including Ag(x) APOB 21-12506.2antigen) MAP4K4--13- 12565 MAP4K4 12565 MAP4K4- 12386.2 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-16- Kinase Kinase Kinase 4(MAP4K4), 12386.2 transcript variant 1 MAP4K4- 12815 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-13- Kinase Kinase Kinase 4(MAP4K4), 12815 transcript variant 1 APOB--13- 12957 NM_000384Apolipoprotein B (including Ag(x) APOB 12957 antigen) MAP4K4--16- 12983Mitogen-Activated Protein Kinase MAP4K4 12983 Kinase Kinase Kinase 4(MAP4K4), transcript variant 1 MAP4K4--16- 12984 Mitogen-ActivatedProtein Kinase MAP4K4 12984 Kinase Kinase Kinase 4 (MAP4K4), transcriptvariant 1 MAP4K4--16- 12985 Mitogen-Activated Protein Kinase MAP4K412985 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1 MAP4K4--16-12986 Mitogen-Activated Protein Kinase MAP4K4 12986 Kinase Kinase Kinase4 (MAP4K4), transcript variant 1 MAP4K4--16- 12987 Mitogen-ActivatedProtein Kinase MAP4K4 12987 Kinase Kinase Kinase 4 (MAP4K4), transcriptvariant 1 MAP4K4--16- 12988 Mitogen-Activated Protein Kinase MAP4K412988 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1 MAP4K4--16-12989 Mitogen-Activated Protein Kinase MAP4K4 12989 Kinase Kinase Kinase4 (MAP4K4), transcript variant 1 MAP4K4--16- 12990 Mitogen-ActivatedProtein Kinase MAP4K4 12990 Kinase Kinase Kinase 4 (MAP4K4), transcriptvariant 1 MAP4K4--16- 12991 Mitogen-Activated Protein Kinase MAP4K412991 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1 MAP4K4--16-12992 Mitogen-Activated Protein Kinase MAP4K4 12992 Kinase Kinase Kinase4 (MAP4K4), transcript variant 1 MAP4K4--16- 12993 Mitogen-ActivatedProtein Kinase MAP4K4 12993 Kinase Kinase Kinase 4 (MAP4K4), transcriptvariant 1 MAP4K4--16- 12994 Mitogen-Activated Protein Kinase MAP4K412994 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1 MAP4K4--16-12995 Mitogen-Activated Protein Kinase MAP4K4 12995 Kinase Kinase Kinase4 (MAP4K4), transcript variant 1 MAP4K4- 13012 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-19- Kinase Kinase Kinase 4(MAP4K4), 13012 transcript variant 1 MAP4K4- 13016 NM_004834Mitogen-Activated Protein Kinase MAP4K4 2931-19- Kinase Kinase Kinase 4(MAP4K4), 13016 transcript variant 1 PPIB--13- 13021 NM_000942Peptidylprolyl Isomerase B PPIB 13021 (cyclophilin B) pGL3-1172- 13038U47296 Cloning vector pGL3-Control pGL3 13-13038 pGL3-1172- 13040 U47296Cloning vector pGL3-Control pGL3 13-13040 --16-13047 13047 SOD1-530-13090 NM_000454 Superoxide Dismutase 1, soluble SOD1 13-13090(amyotrophic lateral sclerosis 1 (adult)) SOD1-523- 13091 NM_000454Superoxide Dismutase 1, soluble SOD1 13-13091 (amyotrophic lateralsclerosis 1 (adult)) SOD1-535- 13092 NM_000454 Superoxide Dismutase 1,soluble SOD1 13-13092 (amyotrophic lateral sclerosis 1 (adult))SOD1-536- 13093 NM_000454 Superoxide Dismutase 1, soluble SOD1 13-13093(amyotrophic lateral sclerosis 1 (adult)) SOD1-396- 13094 NM_000454Superoxide Dismutase 1, soluble SOD1 13-13094 (amyotrophic lateralsclerosis 1 (adult)) SOD1-385- 13095 NM_000454 Superoxide Dismutase 1,soluble SOD1 13-13095 (amyotrophic lateral sclerosis 1 (adult))SOD1-195- 13096 NM_000454 Superoxide Dismutase 1, soluble SOD1 13-13096(amyotrophic lateral sclerosis 1 (adult)) APOB-4314- 13115 NM_000384Apolipoprotein B (including Ag(x) APOB 13-13115 antigen) APOB-3384-13116 NM_000384 Apolipoprotein B (including Ag(x) APOB 13-13116 antigen)APOB-3547- 13117 NM_000384 Apolipoprotein B (including Ag(x) APOB13-13117 antigen) APOB-4318- 13118 NM_000384 Apolipoprotein B (includingAg(x) APOB 13-13118 antigen) APOB-3741- 13119 NM_000384 Apolipoprotein B(including Ag(x) APOB 13-13119 antigen) PPIB--16- 13136 NM_000942Peptidylprolyl Isomerase B PPIB 13136 (cyclophilin B) APOB-4314- 13154NM_000384 Apolipoprotein B (including Ag(x) APOB 15-13154 antigen)APOB-3547- 13155 NM_000384 Apolipoprotein B (including Ag(x) APOB15-13155 antigen) APOB-4318- 13157 NM_000384 Apolipoprotein B (includingAg(x) APOB 15-13157 antigen) APOB-3741- 13158 NM_000384 Apolipoprotein B(including Ag(x) APOB 15-13158 antigen) APOB--13- 13159 NM_000384Apolipoprotein B (including Ag(x) APOB 13159 antigen) APOB--15- 13160NM_000384 Apolipoprotein B (including Ag(x) APOB 13160 antigen)SOD1-530- 13163 NM_000454 Superoxide Dismutase 1, soluble SOD1 16-13163(amyotrophic lateral sclerosis 1 (adult)) SOD1-523- 13164 NM_000454Superoxide Dismutase 1, soluble SOD1 16-13164 (amyotrophic lateralsclerosis 1 (adult)) SOD1-535- 13165 NM_000454 Superoxide Dismutase 1,soluble SOD1 16-13165 (amyotrophic lateral sclerosis 1 (adult))SOD1-536- 13166 NM_000454 Superoxide Dismutase 1, soluble SOD1 16-13166(amyotrophic lateral sclerosis 1 (adult)) SOD1-396- 13167 NM_000454Superoxide Dismutase 1, soluble SOD1 16-13167 (amyotrophic lateralsclerosis 1 (adult)) SOD1-385- 13168 NM_000454 Superoxide Dismutase 1,soluble SOD1 16-13168 (amyotrophic lateral sclerosis 1 (adult))SOD1-195- 13169 NM_000454 Superoxide Dismutase 1, soluble SOD1 16-13169(amyotrophic lateral sclerosis 1 (adult)) pGL3-1172- 13170 U47296Cloning vector pGL3-Control pGL3 16-13170 pGL3-1172- 13171 U47296Cloning vector pGL3-Control pGL3 16-13171 MAP4k4- 13189 NM_004834Mitogen-Activated Protein Kinase MAP4k4 2931-19- Kinase Kinase Kinase 4(MAP4K4), 13189 transcript variant 1 CTGF-1222- 13190 NM_001901.2connective tissue growth factor CTGF 13-13190 CTGF-813- 13192NM_001901.2 connective tissue growth factor CTGF 13-13192 CTGF-747-13194 NM_001901.2 connective tissue growth factor CTGF 13-13194CTGF-817- 13196 NM_001901.2 connective tissue growth factor CTGF13-13196 CTGF-1174- 13198 NM_001901.2 connective tissue growth factorCTGF 13-13198 CTGF-1005- 13200 NM_001901.2 connective tissue growthfactor CTGF 13-13200 CTGF-814- 13202 NM_001901.2 connective tissuegrowth factor CTGF 13-13202 CTGF-816- 13204 NM_001901.2 connectivetissue growth factor CTGF 13-13204 CTGF-1001- 13206 NM_001901.2connective tissue growth factor CTGF 13-13206 CTGF-1173- 13208NM_001901.2 connective tissue growth factor CTGF 13-13208 CTGF-749-13210 NM_001901.2 connective tissue growth factor CTGF 13-13210CTGF-792- 13212 NM_001901.2 connective tissue growth factor CTGF13-13212 CTGF-1162- 13214 NM_001901.2 connective tissue growth factorCTGF 13-13214 CTGF-811- 13216 NM_001901.2 connective tissue growthfactor CTGF 13-13216 CTGF-797- 13218 NM_001901.2 connective tissuegrowth factor CTGF 13-13218 CTGF-1175- 13220 NM_001901.2 connectivetissue growth factor CTGF 13-13220 CTGF-1172- 13222 NM_001901.2connective tissue growth factor CTGF 13-13222 CTGF-1177- 13224NM_001901.2 connective tissue growth factor CTGF 13-13224 CTGF-1176-13226 NM_001901.2 connective tissue growth factor CTGF 13-13226CTGF-812- 13228 NM_001901.2 connective tissue growth factor CTGF13-13228 CTGF-745- 13230 NM_001901.2 connective tissue growth factorCTGF 13-13230 CTGF-1230- 13232 NM_001901.2 connective tissue growthfactor CTGF 13-13232 CTGF-920- 13234 NM_001901.2 connective tissuegrowth factor CTGF 13-13234 CTGF-679- 13236 NM_001901.2 connectivetissue growth factor CTGF 13-13236 CTGF-992- 13238 NM_001901.2connective tissue growth factor CTGF 13-13238 CTGF-1045- 13240NM_001901.2 connective tissue growth factor CTGF 13-13240 CTGF-1231-13242 NM_001901.2 connective tissue growth factor CTGF 13-13242CTGF-991- 13244 NM_001901.2 connective tissue growth factor CTGF13-13244 CTGF-998- 13246 NM_001901.2 connective tissue growth factorCTGF 13-13246 CTGF-1049- 13248 NM_001901.2 connective tissue growthfactor CTGF 13-13248 CTGF-1044- 13250 NM_001901.2 connective tissuegrowth factor CTGF 13-13250 CTGF-1327- 13252 NM_001901.2 connectivetissue growth factor CTGF 13-13252 CTGF-1196- 13254 NM_001901.2connective tissue growth factor CTGF 13-13254 CTGF-562- 13256NM_001901.2 connective tissue growth factor CTGF 13-13256 CTGF-752-13258 NM_001901.2 connective tissue growth factor CTGF 13-13258CTGF-994- 13260 NM_001901.2 connective tissue growth factor CTGF13-13260 CTGF-1040- 13262 NM_001901.2 connective tissue growth factorCTGF 13-13262 CTGF-1984- 13264 NM_001901.2 connective tissue growthfactor CTGF 13-13264 CTGF-2195- 13266 NM_001901.2 connective tissuegrowth factor CTGF 13-13266 CTGF-2043- 13268 NM_001901.2 connectivetissue growth factor CTGF 13-13268 CTGF-1892- 13270 NM_001901.2connective tissue growth factor CTGF 13-13270 CTGF-1567- 13272NM_001901.2 connective tissue growth factor CTGF 13-13272 CTGF-1780-13274 NM_001901.2 connective tissue growth factor CTGF 13-13274CTGF-2162- 13276 NM_001901.2 connective tissue growth factor CTGF13-13276 CTGF-1034- 13278 NM_001901.2 connective tissue growth factorCTGF 13-13278 CTGF-2264- 13280 NM_001901.2 connective tissue growthfactor CTGF 13-13280 CTGF-1032- 13282 NM_001901.2 connective tissuegrowth factor CTGF 13-13282 CTGF-1535- 13284 NM_001901.2 connectivetissue growth factor CTGF 13-13284 CTGF-1694- 13286 NM_001901.2connective tissue growth factor CTGF 13-13286 CTGF-1588- 13288NM_001901.2 connective tissue growth factor CTGF 13-13288 CTGF-928-13290 NM_001901.2 connective tissue growth factor CTGF 13-13290CTGF-1133- 13292 NM_001901.2 connective tissue growth factor CTGF13-13292 CTGF-912- 13294 NM_001901.2 connective tissue growth factorCTGF 13-13294 CTGF-753- 13296 NM_001901.2 connective tissue growthfactor CTGF 13-13296 CTGF-918- 13298 NM_001901.2 connective tissuegrowth factor CTGF 13-13298 CTGF-744- 13300 NM_001901.2 connectivetissue growth factor CTGF 13-13300 CTGF-466- 13302 NM_001901.2connective tissue growth factor CTGF 13-13302 CTGF-917- 13304NM_001901.2 connective tissue growth factor CTGF 13-13304 CTGF-1038-13306 NM_001901.2 connective tissue growth factor CTGF 13-13306CTGF-1048- 13308 NM_001901.2 connective tissue growth factor CTGF13-13308 CTGF-1235- 13310 NM_001901.2 connective tissue growth factorCTGF 13-13310 CTGF-868- 13312 NM_001901.2 connective tissue growthfactor CTGF 13-13312 CTGF-1131- 13314 NM_001901.2 connective tissuegrowth factor CTGF 13-13314 CTGF-1043- 13316 NM_001901.2 connectivetissue growth factor CTGF 13-13316 CTGF-751- 13318 NM_001901.2connective tissue growth factor CTGF 13-13318 CTGF-1227- 13320NM_001901.2 connective tissue growth factor CTGF 13-13320 CTGF-867-13322 NM_001901.2 connective tissue growth factor CTGF 13-13322CTGF-1128- 13324 NM_001901.2 connective tissue growth factor CTGF13-13324 CTGF-756- 13326 NM_001901.2 connective tissue growth factorCTGF 13-13326 CTGF-1234- 13328 NM_001901.2 connective tissue growthfactor CTGF 13-13328 CTGF-916- 13330 NM_001901.2 connective tissuegrowth factor CTGF 13-13330 CTGF-925- 13332 NM_001901.2 connectivetissue growth factor CTGF 13-13332 CTGF-1225- 13334 NM_001901.2connective tissue growth factor CTGF 13-13334 CTGF-445- 13336NM_001901.2 connective tissue growth factor CTGF 13-13336 CTGF-446-13338 NM_001901.2 connective tissue growth factor CTGF 13-13338CTGF-913- 13340 NM_001901.2 connective tissue growth factor CTGF13-13340 CTGF-997- 13342 NM_001901.2 connective tissue growth factorCTGF 13-13342 CTGF-277- 13344 NM_001901.2 connective tissue growthfactor CTGF 13-13344 CTGF-1052- 13346 NM_001901.2 connective tissuegrowth factor CTGF 13-13346 CTGF-887- 13348 NM_001901.2 connectivetissue growth factor CTGF 13-13348 CTGF-914- 13350 NM_001901.2connective tissue growth factor CTGF 13-13350 CTGF-1039- 13352NM_001901.2 connective tissue growth factor CTGF 13-13352 CTGF-754-13354 NM_001901.2 connective tissue growth factor CTGF 13-13354CTGF-1130- 13356 NM_001901.2 connective tissue growth factor CTGF13-13356 CTGF-919- 13358 NM_001901.2 connective tissue growth factorCTGF 13-13358 CTGF-922- 13360 NM_001901.2 connective tissue growthfactor CTGF 13-13360 CTGF-746- 13362 NM_001901.2 connective tissuegrowth factor CTGF 13-13362 CTGF-993- 13364 NM_001901.2 connectivetissue growth factor CTGF 13-13364 CTGF-825- 13366 NM_001901.2connective tissue growth factor CTGF 13-13366 CTGF-926- 13368NM_001901.2 connective tissue growth factor CTGF 13-13368 CTGF-923-13370 NM_001901.2 connective tissue growth factor CTGF 13-13370CTGF-866- 13372 NM_001901.2 connective tissue growth factor CTGF13-13372 CTGF-563- 13374 NM_001901.2 connective tissue growth factorCTGF 13-13374 CTGF-823- 13376 NM_001901.2 connective tissue growthfactor CTGF 13-13376 CTGF-1233- 13378 NM_001901.2 connective tissuegrowth factor CTGF 13-13378 CTGF-924- 13380 NM_001901.2 connectivetissue growth factor CTGF 13-13380 CTGF-921- 13382 NM_001901.2connective tissue growth factor CTGF 13-13382 CTGF-443- 13384NM_001901.2 connective tissue growth factor CTGF 13-13384 CTGF-1041-13386 NM_001901.2 connective tissue growth factor CTGF 13-13386CTGF-1042- 13388 NM_001901.2 connective tissue growth factor CTGF13-13388 CTGF-755- 13390 NM_001901.2 connective tissue growth factorCTGF 13-13390 CTGF-467- 13392 NM_001901.2 connective tissue growthfactor CTGF 13-13392 CTGF-995- 13394 NM_001901.2 connective tissuegrowth factor CTGF 13-13394 CTGF-927- 13396 NM_001901.2 connectivetissue growth factor CTGF 13-13396 SPP1-1025- 13398 NM_000582.2Osteopontin SPP1 13-13398 SPP1-1049- 13400 NM_000582.2 Osteopontin SPP113-13400 SPP1-1051- 13402 NM_000582.2 Osteopontin SPP1 13-13402SPP1-1048- 13404 NM_000582.2 Osteopontin SPP1 13-13404 SPP1-1050- 13406NM_000582.2 Osteopontin SPP1 13-13406 SPP1-1047- 13408 NM_000582.2Osteopontin SPP1 13-13408 SPP1-800- 13410 NM_000582.2 Osteopontin SPP113-13410 SPP1-492- 13412 NM_000582.2 Osteopontin SPP1 13-13412 SPP1-612-13414 NM_000582.2 Osteopontin SPP1 13-13414 SPP1-481- 13416 NM_000582.2Osteopontin SPP1 13-13416 SPP1-614- 13418 NM_000582.2 Osteopontin SPP113-13418 SPP1-951- 13420 NM_000582.2 Osteopontin SPP1 13-13420 SPP1-482-13422 NM_000582.2 Osteopontin SPP1 13-13422 SPP1-856- 13424 NM_000582.2Osteopontin SPP1 13-13424 SPP1-857- 13426 NM_000582.2 Osteopontin SPP113-13426 SPP1-365- 13428 NM_000582.2 Osteopontin SPP1 13-13428 SPP1-359-13430 NM_000582.2 Osteopontin SPP1 13-13430 SPP1-357- 13432 NM_000582.2Osteopontin SPP1 13-13432 SPP1-858- 13434 NM_000582.2 Osteopontin SPP113-13434 SPP1-1012- 13436 NM_000582.2 Osteopontin SPP1 13-13436SPP1-1014- 13438 NM_000582.2 Osteopontin SPP1 13-13438 SPP1-356- 13440NM_000582.2 Osteopontin SPP1 13-13440 SPP1-368- 13442 NM_000582.2Osteopontin SPP1 13-13442 SPP1-1011- 13444 NM_000582.2 Osteopontin SPP113-13444 SPP1-754- 13446 NM_000582.2 Osteopontin SPP1 13-13446SPP1-1021- 13448 NM_000582.2 Osteopontin SPP1 13-13448 SPP1-1330- 13450NM_000582.2 Osteopontin SPP1 13-13450 SPP1-346- 13452 NM_000582.2Osteopontin SPP1 13-13452 SPP1-869- 13454 NM_000582.2 Osteopontin SPP113-13454 SPP1-701- 13456 NM_000582.2 Osteopontin SPP1 13-13456 SPP1-896-13458 NM_000582.2 Osteopontin SPP1 13-13458 SPP1-1035- 13460 NM_000582.2Osteopontin SPP1 13-13460 SPP1-1170- 13462 NM_000582.2 Osteopontin SPP113-13462 SPP1-1282- 13464 NM_000582.2 Osteopontin SPP1 13-13464SPP1-1537- 13466 NM_000582.2 Osteopontin SPP1 13-13466 SPP1-692- 13468NM_000582.2 Osteopontin SPP1 13-13468 SPP1-840- 13470 NM_000582.2Osteopontin SPP1 13-13470 SPP1-1163- 13472 NM_000582.2 Osteopontin SPP113-13472 SPP1-789- 13474 NM_000582.2 Osteopontin SPP1 13-13474 SPP1-841-13476 NM_000582.2 Osteopontin SPP1 13-13476 SPP1-852- 13478 NM_000582.2Osteopontin SPP1 13-13478 SPP1-209- 13480 NM_000582.2 Osteopontin SPP113-13480 SPP1-1276- 13482 NM_000582.2 Osteopontin SPP1 13-13482SPP1-137- 13484 NM_000582.2 Osteopontin SPP1 13-13484 SPP1-711- 13486NM_000582.2 Osteopontin SPP1 13-13486 SPP1-582- 13488 NM_000582.2Osteopontin SPP1 13-13488 SPP1-839- 13490 NM_000582.2 Osteopontin SPP113-13490 SPP1-1091- 13492 NM_000582.2 Osteopontin SPP1 13-13492SPP1-884- 13494 NM_000582.2 Osteopontin SPP1 13-13494 SPP1-903- 13496NM_000582.2 Osteopontin SPP1 13-13496 SPP1-1090- 13498 NM_000582.2Osteopontin SPP1 13-13498 SPP1-474- 13500 NM_000582.2 Osteopontin SPP113-13500 SPP1-575- 13502 NM_000582.2 Osteopontin SPP1 13-13502 SPP1-671-13504 NM_000582.2 Osteopontin SPP1 13-13504 SPP1-924- 13506 NM_000582.2Osteopontin SPP1 13-13506 SPP1-1185- 13508 NM_000582.2 Osteopontin SPP113-13508 SPP1-1221- 13510 NM_000582.2 Osteopontin SPP1 13-13510SPP1-347- 13512 NM_000582.2 Osteopontin SPP1 13-13512 SPP1-634- 13514NM_000582.2 Osteopontin SPP1 13-13514 SPP1-877- 13516 NM_000582.2Osteopontin SPP1 13-13516 SPP1-1033- 13518 NM_000582.2 Osteopontin SPP113-13518 SPP1-714- 13520 NM_000582.2 Osteopontin SPP1 13-13520 SPP1-791-13522 NM_000582.2 Osteopontin SPP1 13-13522 SPP1-813- 13524 NM_000582.2Osteopontin SPP1 13-13524 SPP1-939- 13526 NM_000582.2 Osteopontin SPP113-13526 SPP1-1161- 13528 NM_000582.2 Osteopontin SPP1 13-13528SPP1-1164- 13530 NM_000582.2 Osteopontin SPP1 13-13530 SPP1-1190- 13532NM_000582.2 Osteopontin SPP1 13-13532 SPP1-1333- 13534 NM_000582.2Osteopontin SPP1 13-13534 SPP1-537- 13536 NM_000582.2 Osteopontin SPP113-13536 SPP1-684- 13538 NM_000582.2 Osteopontin SPP1 13-13538 SPP1-707-13540 NM_000582.2 Osteopontin SPP1 13-13540 SPP1-799- 13542 NM_000582.2Osteopontin SPP1 13-13542 SPP1-853- 13544 NM_000582.2 Osteopontin SPP113-13544 SPP1-888- 13546 NM_000582.2 Osteopontin SPP1 13-13546SPP1-1194- 13548 NM_000582.2 Osteopontin SPP1 13-13548 SPP1-1279- 13550NM_000582.2 Osteopontin SPP1 13-13550 SPP1-1300- 13552 NM_000582.2Osteopontin SPP1 13-13552 SPP1-1510- 13554 NM_000582.2 Osteopontin SPP113-13554 SPP1-1543- 13556 NM_000582.2 Osteopontin SPP1 13-13556SPP1-434- 13558 NM_000582.2 Osteopontin SPP1 13-13558 SPP1-600- 13560NM_000582.2 Osteopontin SPP1 13-13560 SPP1-863- 13562 NM_000582.2Osteopontin SPP1 13-13562 SPP1-902- 13564 NM_000582.2 Osteopontin SPP113-13564 SPP1-921- 13566 NM_000582.2 Osteopontin SPP1 13-13566 SPP1-154-13568 NM_000582.2 Osteopontin SPP1 13-13568 SPP1-217- 13570 NM_000582.2Osteopontin SPP1 13-13570 SPP1-816- 13572 NM_000582.2 Osteopontin SPP113-13572 SPP1-882- 13574 NM_000582.2 Osteopontin SPP1 13-13574 SPP1-932-13576 NM_000582.2 Osteopontin SPP1 13-13576 SPP1-1509- 13578 NM_000582.2Osteopontin SPP1 13-13578 SPP1-157- 13580 NM_000582.2 Osteopontin SPP113-13580 SPP1-350- 13582 NM_000582.2 Osteopontin SPP1 13-13582 SPP1-511-13584 NM_000582.2 Osteopontin SPP1 13-13584 SPP1-605- 13586 NM_000582.2Osteopontin SPP1 13-13586 SPP1-811- 13588 NM_000582.2 Osteopontin SPP113-13588 SPP1-892- 13590 NM_000582.2 Osteopontin SPP1 13-13590 SPP1-922-13592 NM_000582.2 Osteopontin SPP1 13-13592 SPP1-1169- 13594 NM_000582.2Osteopontin SPP1 13-13594 SPP1-1182- 13596 NM_000582.2 Osteopontin SPP113-13596 SPP1-1539- 13598 NM_000582.2 Osteopontin SPP1 13-13598SPP1-1541- 13600 NM_000582.2 Osteopontin SPP1 13-13600 SPP1-427- 13602NM_000582.2 Osteopontin SPP1 13-13602 SPP1-533- 13604 NM_000582.2Osteopontin SPP1 13-13604 APOB--13- 13763 NM_000384 Apolipoprotein B(including Ag(x) APOB 13763 antigen) APOB--13- 13764 NM_000384Apolipoprotein B (including Ag(x) APOB 13764 antigen) MAP4K4--16- 13766MAP4K4 13766 PPIB--13- 13767 NM_000942 peptidylprolyl isomerase B PPIB13767 (cyclophilin B) PPIB--15- 13768 NM_000942 peptidylprolyl isomeraseB PPIB 13768 (cyclophilin B) PPIB--17- 13769 NM_000942 peptidylprolylisomerase B PPIB 13769 (cyclophilin B) MAP4K4--16- 13939 MAP4K4 13939APOB-4314- 13940 NM_000384 Apolipoprotein B (including Ag(x) APOB16-13940 antigen) APOB-4314- 13941 NM_000384 Apolipoprotein B (includingAg(x) APOB 17-13941 antigen) APOB--16- 13942 NM_000384 Apolipoprotein B(including Ag(x) APOB 13942 antigen) APOB--18- 13943 NM_000384Apolipoprotein B (including Ag(x) APOB 13943 antigen) APOB--17- 13944NM_000384 Apolipoprotein B (including Ag(x) APOB 13944 antigen)APOB--19- 13945 NM_000384 Apolipoprotein B (including Ag(x) APOB 13945antigen) APOB-4314- 13946 NM_000384 Apolipoprotein B (including Ag(x)APOB 16-13946 antigen) APOB-4314- 13947 NM_000384 Apolipoprotein B(including Ag(x) APOB 17-13947 antigen) APOB--16- 13948 NM_000384Apolipoprotein B (including Ag(x) APOB 13948 antigen) APOB--17- 13949NM_000384 Apolipoprotein B (including Ag(x) APOB 13949 antigen)APOB--16- 13950 NM_000384 Apolipoprotein B (including Ag(x) APOB 13950antigen) APOB--18- 13951 NM_000384 Apolipoprotein B (including Ag(x)APOB 13951 antigen) APOB--17- 13952 NM_000384 Apolipoprotein B(including Ag(x) APOB 13952 antigen) APOB--19- 13953 NM_000384Apolipoprotein B (including Ag(x) APOB 13953 antigen) MAP4K4--16-13766.2 MAP4K4 13766.2 CTGF-1222- 13980 NM_001901.2 connective tissuegrowth factor CTGF 16-13980 CTGF-813- 13981 NM_001901.2 connectivetissue growth factor CTGF 16-13981 CTGF-747- 13982 NM_001901.2connective tissue growth factor CTGF 16-13982 CTGF-817- 13983NM_001901.2 connective tissue growth factor CTGF 16-13983 CTGF-1174-13984 NM_001901.2 connective tissue growth factor CTGF 16-13984CTGF-1005- 13985 NM_001901.2 connective tissue growth factor CTGF16-13985 CTGF-814- 13986 NM_001901.2 connective tissue growth factorCTGF 16-13986 CTGF-816- 13987 NM_001901.2 connective tissue growthfactor CTGF 16-13987 CTGF-1001- 13988 NM_001901.2 connective tissuegrowth factor CTGF 16-13988 CTGF-1173- 13989 NM_001901.2 connectivetissue growth factor CTGF 16-13989 CTGF-749- 13990 NM_001901.2connective tissue growth factor CTGF 16-13990 CTGF-792- 13991NM_001901.2 connective tissue growth factor CTGF 16-13991 CTGF-1162-13992 NM_001901.2 connective tissue growth factor CTGF 16-13992CTGF-811- 13993 NM_001901.2 connective tissue growth factor CTGF16-13993 CTGF-797- 13994 NM_001901.2 connective tissue growth factorCTGF 16-13994 CTGF-1175- 13995 NM_001901.2 connective tissue growthfactor CTGF 16-13995 CTGF-1172- 13996 NM_001901.2 connective tissuegrowth factor CTGF 16-13996 CTGF-1177- 13997 NM_001901.2 connectivetissue growth factor CTGF 16-13997 CTGF-1176- 13998 NM_001901.2connective tissue growth factor CTGF 16-13998 CTGF-812- 13999NM_001901.2 connective tissue growth factor CTGF 16-13999 CTGF-745-14000 NM_001901.2 connective tissue growth factor CTGF 16-14000CTGF-1230- 14001 NM_001901.2 connective tissue growth factor CTGF16-14001 CTGF-920- 14002 NM_001901.2 connective tissue growth factorCTGF 16-14002 CTGF-679- 14003 NM_001901.2 connective tissue growthfactor CTGF 16-14003 CTGF-992- 14004 NM_001901.2 connective tissuegrowth factor CTGF 16-14004 CTGF-1045- 14005 NM_001901.2 connectivetissue growth factor CTGF 16-14005 CTGF-1231- 14006 NM_001901.2connective tissue growth factor CTGF 16-14006 CTGF-991- 14007NM_001901.2 connective tissue growth factor CTGF 16-14007 CTGF-998-14008 NM_001901.2 connective tissue growth factor CTGF 16-14008CTGF-1049- 14009 NM_001901.2 connective tissue growth factor CTGF16-14009 CTGF-1044- 14010 NM_001901.2 connective tissue growth factorCTGF 16-14010 CTGF-1327- 14011 NM_001901.2 connective tissue growthfactor CTGF 16-14011 CTGF-1196- 14012 NM_001901.2 connective tissuegrowth factor CTGF 16-14012 CTGF-562- 14013 NM_001901.2 connectivetissue growth factor CTGF 16-14013 CTGF-752- 14014 NM_001901.2connective tissue growth factor CTGF 16-14014 CTGF-994- 14015NM_001901.2 connective tissue growth factor CTGF 16-14015 CTGF-1040-14016 NM_001901.2 connective tissue growth factor CTGF 16-14016CTGF-1984- 14017 NM_001901.2 connective tissue growth factor CTGF16-14017 CTGF-2195- 14018 NM_001901.2 connective tissue growth factorCTGF 16-14018 CTGF-2043- 14019 NM_001901.2 connective tissue growthfactor CTGF 16-14019 CTGF-1892- 14020 NM_001901.2 connective tissuegrowth factor CTGF 16-14020 CTGF-1567- 14021 NM_001901.2 connectivetissue growth factor CTGF 16-14021 CTGF-1780- 14022 NM_001901.2connective tissue growth factor CTGF 16-14022 CTGF-2162- 14023NM_001901.2 connective tissue growth factor CTGF 16-14023 CTGF-1034-14024 NM_001901.2 connective tissue growth factor CTGF 16-14024CTGF-2264- 14025 NM_001901.2 connective tissue growth factor CTGF16-14025 CTGF-1032- 14026 NM_001901.2 connective tissue growth factorCTGF 16-14026 CTGF-1535- 14027 NM_001901.2 connective tissue growthfactor CTGF 16-14027 CTGF-1694- 14028 NM_001901.2 connective tissuegrowth factor CTGF 16-14028 CTGF-1588- 14029 NM_001901.2 connectivetissue growth factor CTGF 16-14029 CTGF-928- 14030 NM_001901.2connective tissue growth factor CTGF 16-14030 CTGF-1133- 14031NM_001901.2 connective tissue growth factor CTGF 16-14031 CTGF-912-14032 NM_001901.2 connective tissue growth factor CTGF 16-14032CTGF-753- 14033 NM_001901.2 connective tissue growth factor CTGF16-14033 CTGF-918- 14034 NM_001901.2 connective tissue growth factorCTGF 16-14034 CTGF-744- 14035 NM_001901.2 connective tissue growthfactor CTGF 16-14035 CTGF-466- 14036 NM_001901.2 connective tissuegrowth factor CTGF 16-14036 CTGF-917- 14037 NM_001901.2 connectivetissue growth factor CTGF 16-14037 CTGF-1038- 14038 NM_001901.2connective tissue growth factor CTGF 16-14038 CTGF-1048- 14039NM_001901.2 connective tissue growth factor CTGF 16-14039 CTGF-1235-14040 NM_001901.2 connective tissue growth factor CTGF 16-14040CTGF-868- 14041 NM_001901.2 connective tissue growth factor CTGF16-14041 CTGF-1131- 14042 NM_001901.2 connective tissue growth factorCTGF 16-14042 CTGF-1043- 14043 NM_001901.2 connective tissue growthfactor CTGF 16-14043 CTGF-751- 14044 NM_001901.2 connective tissuegrowth factor CTGF 16-14044 CTGF-1227- 14045 NM_001901.2 connectivetissue growth factor CTGF 16-14045 CTGF-867- 14046 NM_001901.2connective tissue growth factor CTGF 16-14046 CTGF-1128- 14047NM_001901.2 connective tissue growth factor CTGF 16-14047 CTGF-756-14048 NM_001901.2 connective tissue growth factor CTGF 16-14048CTGF-1234- 14049 NM_001901.2 connective tissue growth factor CTGF16-14049 CTGF-916- 14050 NM_001901.2 connective tissue growth factorCTGF 16-14050 CTGF-925- 14051 NM_001901.2 connective tissue growthfactor CTGF 16-14051 CTGF-1225- 14052 NM_001901.2 connective tissuegrowth factor CTGF 16-14052 CTGF-445- 14053 NM_001901.2 connectivetissue growth factor CTGF 16-14053 CTGF-446- 14054 NM_001901.2connective tissue growth factor CTGF 16-14054 CTGF-913- 14055NM_001901.2 connective tissue growth factor CTGF 16-14055 CTGF-997-14056 NM_001901.2 connective tissue growth factor CTGF 16-14056CTGF-277- 14057 NM_001901.2 connective tissue growth factor CTGF16-14057 CTGF-1052- 14058 NM_001901.2 connective tissue growth factorCTGF 16-14058 CTGF-887- 14059 NM_001901.2 connective tissue growthfactor CTGF 16-14059 CTGF-914- 14060 NM_001901.2 connective tissuegrowth factor CTGF 16-14060 CTGF-1039- 14061 NM_001901.2 connectivetissue growth factor CTGF 16-14061 CTGF-754- 14062 NM_001901.2connective tissue growth factor CTGF 16-14062 CTGF-1130- 14063NM_001901.2 connective tissue growth factor CTGF 16-14063 CTGF-919-14064 NM_001901.2 connective tissue growth factor CTGF 16-14064CTGF-922- 14065 NM_001901.2 connective tissue growth factor CTGF16-14065 CTGF-746- 14066 NM_001901.2 connective tissue growth factorCTGF 16-14066 CTGF-993- 14067 NM_001901.2 connective tissue growthfactor CTGF 16-14067 CTGF-825- 14068 NM_001901.2 connective tissuegrowth factor CTGF 16-14068 CTGF-926- 14069 NM_001901.2 connectivetissue growth factor CTGF 16-14069 CTGF-923- 14070 NM_001901.2connective tissue growth factor CTGF 16-14070 CTGF-866- 14071NM_001901.2 connective tissue growth factor CTGF 16-14071 CTGF-563-14072 NM_001901.2 connective tissue growth factor CTGF 16-14072CTGF-823- 14073 NM_001901.2 connective tissue growth factor CTGF16-14073 CTGF-1233- 14074 NM_001901.2 connective tissue growth factorCTGF 16-14074 CTGF-924- 14075 NM_001901.2 connective tissue growthfactor CTGF 16-14075 CTGF-921- 14076 NM_001901.2 connective tissuegrowth factor CTGF 16-14076 CTGF-443- 14077 NM_001901.2 connectivetissue growth factor CTGF 16-14077 CTGF-1041- 14078 NM_001901.2connective tissue growth factor CTGF 16-14078 CTGF-1042- 14079NM_001901.2 connective tissue growth factor CTGF 16-14079 CTGF-755-14080 NM_001901.2 connective tissue growth factor CTGF 16-14080CTGF-467- 14081 NM_001901.2 connective tissue growth factor CTGF16-14081 CTGF-995- 14082 NM_001901.2 connective tissue growth factorCTGF 16-14082 CTGF-927- 14083 NM_001901.2 connective tissue growthfactor CTGF 16-14083 SPP1-1091- 14131 NM_000582.2 Osteopontin SPP116-14131 PPIB--16- 14188 NM_000942 peptidylprolyl isomerase B PPIB 14188(cyclophilin B) PPIB--17- 14189 NM_000942 peptidylprolyl isomerase BPPIB 14189 (cyclophilin B) PPIB--18- 14190 NM_000942 peptidylprolylisomerase B PPIB 14190 (cyclophilin B) pGL3-1172- 14386 U47296 Cloningvector pGL3-Control pGL3 16-14386 pGL3-1172- 14387 U47296 Cloning vectorpGL3-Control pGL3 16-14387 MAP4K4- 14390 NM_004834 Mitogen-ActivatedProtein Kinase MAP4K4 2931-25- Kinase Kinase Kinase 4 (MAP4K4), 14390transcript variant 1 miR-122-- 14391 miR- 23-14391 122 14084 NM_000582.2Osteopontin SPP1 14085 NM_000582.2 Osteopontin SPP1 14086 NM_000582.2Osteopontin SPP1 14087 NM_000582.2 Osteopontin SPP1 14088 NM_000582.2Osteopontin SPP1 14089 NM_000582.2 Osteopontin SPP1 14090 NM_000582.2Osteopontin SPP1 14091 NM_000582.2 Osteopontin SPP1 14092 NM_000582.2Osteopontin SPP1 14093 NM_000582.2 Osteopontin SPP1 14094 NM_000582.2Osteopontin SPP1 14095 NM_000582.2 Osteopontin SPP1 14096 NM_000582.2Osteopontin SPP1 14097 NM_000582.2 Osteopontin SPP1 14098 NM_000582.2Osteopontin SPP1 14099 NM_000582.2 Osteopontin SPP1 14100 NM_000582.2Osteopontin SPP1 14101 NM_000582.2 Osteopontin SPP1 14102 NM_000582.2Osteopontin SPP1 14103 NM_000582.2 Osteopontin SPP1 14104 NM_000582.2Osteopontin SPP1 14105 NM_000582.2 Osteopontin SPP1 14106 NM_000582.2Osteopontin SPP1 14107 NM_000582.2 Osteopontin SPP1 14108 NM_000582.2Osteopontin SPP1 14109 NM_000582.2 Osteopontin SPP1 14110 NM_000582.2Osteopontin SPP1 14111 NM_000582.2 Osteopontin SPP1 14112 NM_000582.2Osteopontin SPP1 14113 NM_000582.2 Osteopontin SPP1 14114 NM_000582.2Osteopontin SPP1 14115 NM_000582.2 Osteopontin SPP1 14116 NM_000582.2Osteopontin SPP1 14117 NM_000582.2 Osteopontin SPP1 14118 NM_000582.2Osteopontin SPP1 14119 NM_000582.2 Osteopontin SPP1 14120 NM_000582.2Osteopontin SPP1 14121 NM_000582.2 Osteopontin SPP1 14122 NM_000582.2Osteopontin SPP1 14123 NM_000582.2 Osteopontin SPP1 14124 NM_000582.2Osteopontin SPP1 14125 NM_000582.2 Osteopontin SPP1 14126 NM_000582.2Osteopontin SPP1 14127 NM_000582.2 Osteopontin SPP1 14128 NM_000582.2Osteopontin SPP1 14129 NM_000582.2 Osteopontin SPP1 14130 NM_000582.2Osteopontin SPP1 14132 NM_000582.2 Osteopontin SPP1 14133 NM_000582.2Osteopontin SPP1 14134 NM_000582.2 Osteopontin SPP1 14135 NM_000582.2Osteopontin SPP1 14136 NM_000582.2 Osteopontin SPP1 14137 NM_000582.2Osteopontin SPP1 14138 NM_000582.2 Osteopontin SPP1 14139 NM_000582.2Osteopontin SPP1 14140 NM_000582.2 Osteopontin SPP1 14141 NM_000582.2Osteopontin SPP1 14142 NM_000582.2 Osteopontin SPP1 14143 NM_000582.2Osteopontin SPP1 14144 NM_000582.2 Osteopontin SPP1 14145 NM_000582.2Osteopontin SPP1 14146 NM_000582.2 Osteopontin SPP1 14147 NM_000582.2Osteopontin SPP1 14148 NM_000582.2 Osteopontin SPP1 14149 NM_000582.2Osteopontin SPP1 14150 NM_000582.2 Osteopontin SPP1 14151 NM_000582.2Osteopontin SPP1 14152 NM_000582.2 Osteopontin SPP1 14153 NM_000582.2Osteopontin SPP1 14154 NM_000582.2 Osteopontin SPP1 14155 NM_000582.2Osteopontin SPP1 14156 NM_000582.2 Osteopontin SPP1 14157 NM_000582.2Osteopontin SPP1 14158 NM_000582.2 Osteopontin SPP1 14159 NM_000582.2Osteopontin SPP1 14160 NM_000582.2 Osteopontin SPP1 14161 NM_000582.2Osteopontin SPP1 14162 NM_000582.2 Osteopontin SPP1 14163 NM_000582.2Osteopontin SPP1 14164 NM_000582.2 Osteopontin SPP1 14165 NM_000582.2Osteopontin SPP1 14166 NM_000582.2 Osteopontin SPP1 14167 NM_000582.2Osteopontin SPP1 14168 NM_000582.2 Osteopontin SPP1 14169 NM_000582.2Osteopontin SPP1 14170 NM_000582.2 Osteopontin SPP1 14171 NM_000582.2Osteopontin SPP1 14172 NM_000582.2 Osteopontin SPP1 14173 NM_000582.2Osteopontin SPP1 14174 NM_000582.2 Osteopontin SPP1 14175 NM_000582.2Osteopontin SPP1 14176 NM_000582.2 Osteopontin SPP1 14177 NM_000582.2Osteopontin SPP1 14178 NM_000582.2 Osteopontin SPP1 14179 NM_000582.2Osteopontin SPP1 14180 NM_000582.2 Osteopontin SPP1 14181 NM_000582.2Osteopontin SPP1 14182 NM_000582.2 Osteopontin SPP1 14183 NM_000582.2Osteopontin SPP1 14184 NM_000582.2 Osteopontin SPP1 14185 NM_000582.2Osteopontin SPP1 14186 NM_000582.2 Osteopontin SPP1 14187 NM_000582.2Osteopontin SPP1

TABLE 2Antisense backbone, chemistry, and sequence information. o: phosphodiester;s: phosphorothioate; P: 5′phosphorylation; 0: 2′-OH; F: 2′-fluoro;m: 2′O-methyl; +: LNA modification. Capital letters in the sequencesignify riobonucleotides, lower case letters signify deoxyribonucleotides.Oligo AntiSense AntiSense AntiSense SEQ ID ID Number Number BackboneChemistry Sequence NO: APOB- 12138 ooooooooooooo 00000000000000AUUGGUAUUCAGUGUGA 1 10167-20- oooooo 000000m UG 12138 APOB- 12139ooooooooooooo 00000000000000 AUUCGUAUUGAGUCUGA 2 10167-20- oooooo000000m UC 12139 MAP4K4- 12266 2931-13- 12266 MAP4K4- 12293ooooooooooooo Pf000fffff0f00 UAGACUUCCACAGAACU 3 2931-16- oooooo 00fff0CU 12293 MAP4K4- 12383 ooooooooooooo 00000000000000 UAGACUUCCACAGAACU 42931-16- oooooo 00000 CU 12383 MAP4K4- 12384 oooooooooooooP0000000000000 UAGACUUCCACAGAACU 5 2931-16- oooooo 000000 CU 12384MAP4K4- 12385 ooooooooooooo Pf000fffff0f00 UAGACUUCCACAGAACU 6 2931-16-oooooo 00fff0 CU 12385 MAP4K4- 12386 oooooooooosss Pf000fffff0f00UAGACUUCCACAGAACU 7 2931-16- ssssso 00fff0 CU 12386 MAP4K4- 12387oooooooooosss P0000000000000 UAGACUUCCACAGAACU 8 2931-16- ssssso 000000CU 12387 MAP4K4- 12388 ooooooooooooo 00000000000000 UAGACUUCCACAGAACU 92931-15- oooo 000 12388 MAP4K4- 12432 2931-13- 12432 MAP4K4- 12266.22931-13- 12266.2 APOB--21- 12434 ooooooooooooo 00000000000000AUUGGUAUUCAGUGUGA 10 12434 oooooooo 000000m UGAC APOB--21- 12435ooooooooooooo 00000000000000 AUUCGUAUUGAGUCUGA 11 12435 oooooooo 000000mUCAC MAP4K4- 12451 oooooooooosss Pf000fffff0f00 UAGACUUCCACAGAACU 122931-16- ssssso 00ffmm CU 12451 MAP4K4- 12452 oooooooooosssPm000fffff0f00 UAGACUUCCACAGAACU 13 2931-16- ssssso 00ffmm CU 12452MAP4K4- 12453 oooooosssssss Pm000fffff0f00 UAGACUUCCACAGAACU 14 2931-16-ssssso 00ffmm CU 12453 MAP4K4- 12454 oooooooooooos Pm000fffff0f00UAGACUUCCACAGAACU 15 2931-17- ssssssso 00ffffmm CUUC 12454 MAP4K4- 12455oooooooosssss Pm000fffff0f00 UAGACUUCCACAGAACU 16 2931-17- ssssssso00ffffmm CUUC 12455 MAP4K4- 12456 oooooooooooos Pm000fffff0f00UAGACUUCCACAGAACU 17 2931-19- ssssssssssso 00ffffff00mm CUUCAAAG 12456--27-12480 12480 --27-12481 12481 APOB- 12505 ooooooooooooo00000000000000 AUUGGUAUUCAGUGUGA 18 10167-21- ooooooos 000000m UGAC12505 APOB- 12506 ooooooooooooo 00000000000000 AUUCGUAUUGAGUCUGA 1910167-21- ooooooos 000000m UCAC 12506 MAP4K4- 12539 ooooooooooossPf000fffff0f00 UAGACUUCCACAGAACU 20 2931-16- ssssss 00fff0 CU 12539APOB- 12505.2 ooooooooooooo 00000000000000 AUUGGUAUUCAGUGUGA 2110167-21- oooooooo 000000m UGAC 12505.2 APOB- 12506.2 ooooooooooooo00000000000000 AUUCGUAUUGAGUCUGA 22 10167-21- oooooooo 000000m UCAC12506.2 MAP4K4-- 12565 13-12565 MAP4K4- 12386.2 oooooooooosssPf000fffff0f00 UAGACUUCCACAGAACU 23 2931-16- ssssso 00fff0 CU 12386.2MAP4K4- 12815 2931-13- 12815 APOB--13- 12957 12957 MAP4K4-- 12983oooooooooooos Pm000fffff0m00 uagacuuccacagaacu 24 16-12983 ssssso 00mmm0cu MAP4K4-- 12984 oooooooooooos Pm000fffff0m00 uagacuuccacagaacu 2516-12984 sssss 00mmm0 cu MAP4K4-- 12985 oooooooooooos Pm000fffff0m00uagacuuccacagaacu 26 16-12985 ssssso 00mmm0 cu MAP4K4-- 12986oooooooooosss Pf000fffff0f00 UAGACUUCCACAGAACU 27 16-12986 ssssso 00fff0CU MAP4K4-- 12987 ooooooooooooo P0000f00ff0m00 UagacUUccacagaacU 2816-12987 ssssss 00m0m0 cU MAP4K4-- 12988 ooooooooooooo P0000f00ff0m00UagacUUccacagaacU 29 16-12988 ssssss 00m0m0 cu MAP4K4-- 12989ooooooooooooo P0000ff0ff0m00 UagacuUccacagaacU 30 16-12989 ssssss 00m0m0cu MAP4K4-- 12990 ooooooooooooo Pf0000ff000000 uagaCuuCCaCagaaCu 3116-12990 ssssss 000m00 Cu MAP4K4-- 12991 ooooooooooooo Pf0000fff00m00uagaCuucCacagaaCu 32 16-12991 ssssss 000mm0 cu MAP4K4-- 12992ooooooooooooo Pf000fffff0000 uagacuuccaCagaaCu 33 16-12992 ssssss 000m00Cu MAP4K4-- 12993 ooooooooooooo P0000000000000 UagaCUUCCaCagaaCU 3416-12993 ssssss 000000 CU MAP4K4-- 12994 ooooooooooooo P0000f0f0f0000UagacUuCcaCagaaCu 35 16-12994 ssssss 000m00 Cu MAP4K4-- 12995oooooooooooos Pf000fffff0000 uagacuuccaCagaaCU 36 16-12995 ssssso 000000CU MAP4K4- 13012 2931-19- 13012 MAP4K4- 13016 2931-19- 13016 PPIB--13-13021 13021 pGL3-1172- 13038 13-13038 pGL3-1172- 13040 13-13040--16-13047 13047 oooooooooooos Pm000000000m00 UAGACUUCCACAGAACU 37 sssss00mmm0 CU SOD1-530- 13090 13-13090 SOD1-523- 13091 13-13091 SOD1-535-13092 13-13092 SOD1-536- 13093 13-13093 SOD1-396- 13094 13-13094SOD1-385- 13095 13-13095 SOD1-195- 13096 13-13096 APOB-4314- 1311513-13115 APOB-3384- 13116 13-13116 APOB-3547- 13117 13-13117 APOB-4318-13118 13-13118 APOB-3741- 13119 13-13119 PPIB--16- 13136 oooooooooooosPm0fffff0f00mm UGUUUUUGUAGCCAAAU 38 13136 sssss 000mm0 CC APOB-4314-13154 15-13154 APOB-3547- 13155 15-13155 APOB-4318- 13157 15-13157APOB-3741- 13158 15-13158 APOB--13- 13159 13159 APOB--15- 13160 13160SOD1-530- 13163 oooooooooooos Pm0ffffffff0mm UACUUUCUUCAUUUCCA 3916-13163 ssssso mmm0m0 CC SOD1-523- 13164 oooooooooooos Pmff0fffff0fmmUUCAUUUCCACCUUUGC 40 16-13164 ssssso mm0mm0 CC SOD1-535- 13165oooooooooooos Pmfff0f0ffffmm CUUUGUACUUUCUUCAU 41 16-13165 ssssso mm0mm0UU SOD1-536- 13166 oooooooooooos Pmffff0f0fffmm UCUUUGUACUUUCUUCA 4216-13166 ssssso mmm0m0 UU SOD1-396- 13167 oooooooooooos Pmf00f00ff0f0mUCAGCAGUCACAUUGCC 43 16-13167 ssssso m0mmm0 CA SOD1-385- 13168oooooooooooos Pmff0fff000fmm AUUGCCCAAGUCUCCAA 44 16-13168 ssssso mm00m0CA SOD1-195- 13169 oooooooooooos Pmfff0fff0000m UUCUGCUCGAAAUUGAU 4516-13169 ssssso m00m00 GA pGL3-1172- 13170 oooooooooooos Pm00ff0f0ffm0fAAAUCGUAUUUGUCAAU 46 16-13170 ssssso f00mm0 CA pGL3-1172- 13171ooooooooooooo Pm00ff0f0ffm0f AAAUCGUAUUUGUCAAU 47 16-13171 ssssss f00mm0CA MAP4k4- 13189 ooooooooooooo 00000000000000 UAGACUUCCACAGAACU 482931-19- oooooo 00000 CU 13189 CTGF-1222- 13190 13-13190 CTGF-813- 1319213-13192 CTGF-747- 13194 13-13194 CTGF-817- 13196 13-13196 CTGF-1174-13198 13-13198 CTGF-1005- 13200 13-13200 CTGF-814- 13202 13-13202CTGF-816- 13204 13-13204 CTGF-1001- 13206 13-13206 CTGF-1173- 1320813-13208 CTGF-749- 13210 13-13210 CTGF-792- 13212 13-13212 CTGF-1162-13214 13-13214 CTGF-811- 13216 13-13216 CTGF-797- 13218 13-13218CTGF-1175- 13220 13-13220 CTGF-1172- 13222 13-13222 CTGF-1177- 1322413-13224 CTGF-1176- 13226 13-13226 CTGF-812- 13228 13-13228 CTGF-745-13230 13-13230 CTGF-1230- 13232 13-13232 CTGF-920- 13234 13-13234CTGF-679- 13236 13-13236 CTGF-992- 13238 13-13238 CTGF-1045- 1324013-13240 CTGF-1231- 13242 13-13242 CTGF-991- 13244 13-13244 CTGF-998-13246 13-13246 CTGF-1049- 13248 13-13248 CTGF-1044- 13250 13-13250CTGF-1327- 13252 13-13252 CTGF-1196- 13254 13-13254 CTGF-562- 1325613-13256 CTGF-752- 13258 13-13258 CTGF-994- 13260 13-13260 CTGF-1040-13262 13-13262 CTGF-1984- 13264 13-13264 CTGF-2195- 13266 13-13266CTGF-2043- 13268 13-13268 CTGF-1892- 13270 13-13270 CTGF-1567- 1327213-13272 CTGF-1780- 13274 13-13274 CTGF-2162- 13276 13-13276 CTGF-1034-13278 13-13278 CTGF-2264- 13280 13-13280 CTGF-1032- 13282 13-13282CTGF-1535- 13284 13-13284 CTGF-1694- 13286 13-13286 CTGF-1588- 1328813-13288 CTGF-928- 13290 13-13290 CTGF-1133- 13292 13-13292 CTGF-912-13294 13-13294 CTGF-753- 13296 13-13296 CTGF-918- 13298 13-13298CTGF-744- 13300 13-13300 CTGF-466- 13302 13-13302 CTGF-917- 1330413-13304 CTGF-1038- 13306 13-13306 CTGF-1048- 13308 13-13308 CTGF-1235-13310 13-13310 CTGF-868- 13312 13-13312 CTGF-1131- 13314 13-13314CTGF-1043- 13316 13-13316 CTGF-751- 13318 13-13318 CTGF-1227- 1332013-13320 CTGF-867- 13322 13-13322 CTGF-1128- 13324 13-13324 CTGF-756-13326 13-13326 CTGF-1234- 13328 13-13328 CTGF-916- 13330 13-13330CTGF-925- 13332 13-13332 CTGF-1225- 13334 13-13334 CTGF-445- 1333613-13336 CTGF-446- 13338 13-13338 CTGF-913- 13340 13-13340 CTGF-997-13342 13-13342 CTGF-277- 13344 13-13344 CTGF-1052- 13346 13-13346CTGF-887- 13348 13-13348 CTGF-914- 13350 13-13350 CTGF-1039- 1335213-13352 CTGF-754- 13354 13-13354 CTGF-1130- 13356 13-13356 CTGF-919-13358 13-13358 CTGF-922- 13360 13-13360 CTGF-746- 13362 13-13362CTGF-993- 13364 13-13364 CTGF-825- 13366 13-13366 CTGF-926- 1336813-13368 CTGF-923- 13370 13-13370 CTGF-866- 13372 13-13372 CTGF-563-13374 13-13374 CTGF-823- 13376 13-13376 CTGF-1233- 13378 13-13378CTGF-924- 13380 13-13380 CTGF-921- 13382 13-13382 CTGF-443- 1338413-13384 CTGF-1041- 13386 13-13386 CTGF-1042- 13388 13-13388 CTGF-755-13390 13-13390 CTGF-467- 13392 13-13392 CTGF-995- 13394 13-13394CTGF-927- 13396 13-13396 SPP1-1025- 13398 13-13398 SPP1-1049- 1340013-13400 SPP1-1051- 13402 13-13402 SPP1-1048- 13404 13-13404 SPP1-1050-13406 13-13406 SPP1-1047- 13408 13-13408 SPP1-800- 13410 13-13410SPP1-492- 13412 13-13412 SPP1-612- 13414 13-13414 SPP1-481- 1341613-13416 SPP1-614- 13418 13-13418 SPP1-951- 13420 13-13420 SPP1-482-13422 13-13422 SPP1-856- 13424 13-13424 SPP1-857- 13426 13-13426SPP1-365- 13428 13-13428 SPP1-359- 13430 13-13430 SPP1-357- 1343213-13432 SPP1-858- 13434 13-13434 SPP1-1012- 13436 13-13436 SPP1-1014-13438 13-13438 SPP1-356- 13440 13-13440 SPP1-368- 13442 13-13442SPP1-1011- 13444 13-13444 SPP1-754- 13446 13-13446 SPP1-1021- 1344813-13448 SPP1-1330- 13450 13-13450 SPP1-346- 13452 13-13452 SPP1-869-13454 13-13454 SPP1-701- 13456 13-13456 SPP1-896- 13458 13-13458SPP1-1035- 13460 13-13460 SPP1-1170- 13462 13-13462 SPP1-1282- 1346413-13464 SPP1-1537- 13466 13-13466 SPP1-692- 13468 13-13468 SPP1-840-13470 13-13470 SPP1-1163- 13472 13-13472 SPP1-789- 13474 13-13474SPP1-841- 13476 13-13476 SPP1-852- 13478 13-13478 SPP1-209- 1348013-13480 SPP1-1276- 13482 13-13482 SPP1-137- 13484 13-13484 SPP1-711-13486 13-13486 SPP1-582- 13488 13-13488 SPP1-839- 13490 13-13490SPP1-1091- 13492 13-13492 SPP1-884- 13494 13-13494 SPP1-903- 1349613-13496 SPP1-1090- 13498 13-13498 SPP1-474- 13500 13-13500 SPP1-575-13502 13-13502 SPP1-671- 13504 13-13504 SPP1-924- 13506 13-13506SPP1-1185- 13508 13-13508 SPP1-1221- 13510 13-13510 SPP1-347- 1351213-13512 SPP1-634- 13514 13-13514 SPP1-877- 13516 13-13516 SPP1-1033-13518 13-13518 SPP1-714- 13520 13-13520 SPP1-791- 13522 13-13522SPP1-813- 13524 13-13524 SPP1-939- 13526 13-13526 SPP1-1161- 1352813-13528 SPP1-1164- 13530 13-13530 SPP1-1190- 13532 13-13532 SPP1-1333-13534 13-13534 SPP1-537- 13536 13-13536 SPP1-684- 13538 13-13538SPP1-707- 13540 13-13540 SPP1-799- 13542 13-13542 SPP1-853- 1354413-13544 SPP1-888- 13546 13-13546 SPP1-1194- 13548 13-13548 SPP1-1279-13550 13-13550 SPP1-1300- 13552 13-13552 SPP1-1510- 13554 13-13554SPP1-1543- 13556 13-13556 SPP1-434- 13558 13-13558 SPP1-600- 1356013-13560 SPP1-863- 13562 13-13562 SPP1-902- 13564 13-13564 SPP1-921-13566 13-13566 SPP1-154- 13568 13-13568 SPP1-217- 13570 13-13570SPP1-816- 13572 13-13572 SPP1-882- 13574 13-13574 SPP1-932- 1357613-13576 SPP1-1509- 13578 13-13578 SPP1-157- 13580 13-13580 SPP1-350-13582 13-13582 SPP1-511- 13584 13-13584 SPP1-605- 13586 13-13586SPP1-811- 13588 13-13588 SPP1-892- 13590 13-13590 SPP1-922- 1359213-13592 SPP1-1169- 13594 13-13594 SPP1-1182- 13596 13-13596 SPP1-1539-13598 13-13598 SPP1-1541- 13600 13-13600 SPP1-427- 13602 13-13602SPP1-533- 13604 13-13604 APOB--13- 13763 13763 APOB--13- 13764 13764MAP4K4-- 13766 oooooooooooos Pm000fffff0m00 UAGACUUCCACAGAACU 4916-13766 ssssso 00mmm0 CU PPIB--13- 13767 13767 PPIB--15- 13768 13768PPIB--17- 13769 13769 MAP4K4-- 13939 oooooooooooos m000f0ffff0m0mUAGACAUCCUACACAGC 50 16-13939 ssssso 00m0m AC APOB-4314- 13940oooooooooooos Pm0fffffff000m UGUUUCUCCAGAUCCUU 51 16-13940 ssssso mmmm00GC APOB-4314- 13941 oooooooooooos Pm0fffffff000m UGUUUCUCCAGAUCCUU 5217-13941 ssssso mmmm00 GC APOB--16- 13942 oooooooooooos Pm00f000f000mmUAGCAGAUGAGUCCAUU 53 13942 ssssso m0mmm0 UG APOB--18- 13943ooooooooooooo Pm00f000f000mm UAGCAGAUGAGUCCAUU 54 13943 ooossssssom0mmm00000 UGGAGA APOB--17- 13944 oooooooooooos Pm00f000f000mmUAGCAGAUGAGUCCAUU 55 13944 ssssso m0mmm0 UG APOB--19- 13945ooooooooooooo Pm00f000f000mm UAGCAGAUGAGUCCAUU 56 13945 ooossssssom0mmm00000 UGGAGA APOB-4314- 13946 oooooooooooos Pmf0ff0ffffmmmAUGUUGUUUCUCCAGAU 57 16-13946 ssssso 000mm0 CC APOB-4314- 13947oooooooooooos Pmf0ff0ffffmmm AUGUUGUUUCUCCAGAU 58 17-13947 ssssso 000mm0CC APOB--16- 13948 oooooooooooos Pm0fff000000mm UGUUUGAGGGACUCUGU 5913948 ssssso mm0m00 GA APOB--17- 13949 oooooooooooos Pm0fff000000mmUGUUUGAGGGACUCUGU 60 13949 ssssso mm0m00 GA APOB--16- 13950oooooooooooos Pmff00f0fff00m AUUGGUAUUCAGUGUGA 61 13950 ssssso 0m00m0 UGAPOB--18- 13951 ooooooooooooo Pmff00f0fff00m AUUGGUAUUCAGUGUGA 62 13951ooosssssso 0m00m00m00 UGACAC APOB--17- 13952 oooooooooooosPmff00f0fff00m AUUGGUAUUCAGUGUGA 63 13952 ssssso 0m00m0 UG APOB--19-13953 ooooooooooooo Pmff00f0fff00m AUUGGUAUUCAGUGUGA 64 13953 ooosssssso0m00m00m00 UGACAC MAP4K4-- 13766.2 oooooooooooos Pm000fffff0m00UAGACUUCCACAGAACU 65 16-13766.2 ssssso 00mmm0 CU CTGF-1222- 13980oooooooooooos Pm0f0ffffffm0m UACAUCUUCCUGUAGUA 66 16-13980 ssssso 00m0m0CA CTGF-813- 13981 oooooooooooos Pm0f0ffff0mmmm AGGCGCUCCACUCUGUG 6716-13981 ssssso 0m000 GU CTGF-747- 13982 oooooooooooos Pm0ffffff00mm0UGUCUUCCAGUCGGUAA 68 16-13982 ssssso m0000 GC CTGF-817- 13983oooooooooooos Pm00f000f0fmmm GAACAGGCGCUCCACUC 69 16-13983 ssssso 0mmmm0UG CTGF-1174- 13984 oooooooooooos Pm00ff0f00f00m CAGUUGUAAUGGCAGGC 7016-13984 ssssso 000m00 AC CTGF-1005- 13985 oooooooooooos Pmff000000mmm0AGCCAGAAAGCUCAAAC 71 16-13985 ssssso 00mm0 UU CTGF-814- 13986oooooooooooos Pm000f0ffff0mm CAGGCGCUCCACUCUGU 72 16-13986 ssssso mm0m00GG CTGF-816- 13987 oooooooooooos Pm0f000f0ffmm0 AACAGGCGCUCCACUCU 7316-13987 ssssso mmmm00 GU CTGF-1001- 13988 oooooooooooos Pm0000fff000mmAGAAAGCUCAAACUUGA 74 16-13988 ssssso m00m0 UA CTGF-1173- 13989oooooooooooos Pmff0f00f00m00 AGUUGUAAUGGCAGGCA 75 16-13989 ssssso 0m0m0CA CTGF-749- 13990 oooooooooooos Pmf0ffffff00mm CGUGUCUUCCAGUCGGU 7616-13990 ssssso 00m00 AA CTGF-792- 13991 oooooooooooos Pm00ff000f00mmGGACCAGGCAGUUGGCU 77 16-13991 ssssso 00mmm0 CU CTGF-1162- 13992oooooooooooos Pm000f0f000mmm CAGGCACAGGUCUUGAU 78 16-13992 ssssso m00m00GA CTGF-811- 13993 oooooooooooos Pmf0ffff0ffmm0 GCGCUCCACUCUGUGGU 7916-13993 ssssso m00mm0 CU CTGF-797- 13994 oooooooooooos Pm0fff000ff000GGUCUGGACCAGGCAGU 80 16-13994 ssssso m00mm0 UG CTGF-1175- 13995oooooooooooos Pmf00ff0f00m00 ACAGUUGUAAUGGCAGG 81 16-13995 ssssso m000m0CA CTGF-1172- 13996 oooooooooooos Pmff0f00f00m00 GUUGUAAUGGCAGGCAC 8216-13996 ssssso 0m0m00 AG CTGF-1177- 13997 oooooooooooos Pm00f00ff0f00mGGACAGUUGUAAUGGCA 83 16-13997 ssssso 00m000 GG CTGF-1176- 13998oooooooooooos Pm0f00ff0f00m0 GACAGUUGUAAUGGCAG 84 16-13998 ssssso 0m0000GC CTGF-812- 13999 oooooooooooos Pm0f0ffff0fmmm GGCGCUCCACUCUGUGG 8516-13999 ssssso 0m00m0 UC CTGF-745- 14000 oooooooooooos Pmfffff00ff00mUCUUCCAGUCGGUAAGC 86 16-14000 ssssso 000mm0 CG CTGF-1230- 14001oooooooooooos Pm0fffff0f0m0m UGUCUCCGUACAUCUUC 87 16-14001 ssssso mmmmm0CU CTGF-920- 14002 oooooooooooos Pmffff0f0000mm AGCUUCGCAAGGCCUGA 8816-14002 ssssso m00m0 CC CTGF-679- 14003 oooooooooooos Pm0ffffff0f00mCACUCCUCGCAGCAUUU 89 16-14003 ssssso 0mmmm0 CC CTGF-992- 14004oooooooooooos Pm00fff00f000m AAACUUGAUAGGCUUGG 90 16-14004 ssssso mm0000AG CTGF-1045- 14005 oooooooooooos Pmffff0f0000mm ACUCCACAGAAUUUAGC 9116-14005 ssssso m00mm0 UC CTGF-1231- 14006 oooooooooooos Pmf0fffff0f0m0AUGUCUCCGUACAUCUU 92 16-14006 ssssso mmmmm0 CC CTGF-991- 14007oooooooooooos Pm0fff00f000mm AACUUGAUAGGCUUGGA 93 16-14007 ssssso m00000GA CTGF-998- 14008 oooooooooooos Pm00fff000fmm0 AAGCUCAAACUUGAUAG 9416-14008 ssssso 0m0000 GC CTGF-1049- 14009 oooooooooooos Pmf0f0ffff0m00ACAUACUCCACAGAAUU 95 16-14009 ssssso 00mmm0 UA CTGF-1044- 14010oooooooooooos Pmfff0f0000mmm CUCCACAGAAUUUAGCU 96 16-14010 ssssso 00mmm0CG CTGF-1327- 14011 oooooooooooos Pm0f0ff0ff0000 UGUGCUACUGAAAUCAU 9716-14011 ssssso mm0mm0 UU CTGF-1196- 14012 oooooooooooos Pm0000f0ff0mm0AAAGAUGUCAUUGUCUC 98 16-14012 ssssso mmmmm0 CG CTGF-562- 14013oooooooooooos Pmf0f0ff00f0mm GUGCACUGGUACUUGCA 99 16-14013 ssssso m0m000GC CTGF-752- 14014 oooooooooooos Pm00f0f0fffmmm AAACGUGUCUUCCAGUC 10016-14014 ssssso 00mm00 GG CTGF-994- 14015 oooooooooooos Pmf000fff00m00UCAAACUUGAUAGGCUU 101 16-14015 ssssso 0mmm00 GG CTGF-1040- 14016oooooooooooos Pmf0000fff00mm ACAGAAUUUAGCUCGGU 102 16-14016 sssssom00m00 AU CTGF-1984- 14017 oooooooooooos Pmf0f0ffff0mmmUUACAUUCUACCUAUGG 103 16-14017 ssssso 0m00m0 UG CTGF-2195- 14018oooooooooooos Pm00ff00ff00mm AAACUGAUCAGCUAUAU 104 16-14018 ssssso0m0m00 AG CTGF-2043- 14019 oooooooooooos Pm0fff000f0000UAUCUGAGCAGAAUUUC 105 16-14019 ssssso mmmmm0 CA CTGF-1892- 14020oooooooooooos Pmf00fff000m00 UUAACUUAGAUAACUGU 106 16-14020 sssssomm0m00 AC CTGF-1567- 14021 oooooooooooos Pm0ff0fff0f0m0UAUUACUCGUAUAAGAU 107 16-14021 ssssso 000m00 GC CTGF-1780- 14022oooooooooooos Pm00ff0fff00mm AAGCUGUCCAGUCUAAU 108 16-14022 sssssom00mm0 CG CTGF-2162- 14023 oooooooooooos Pm00f00000fm0mUAAUAAAGGCCAUUUGU 109 16-14023 ssssso mm0mm0 UC CTGF-1034- 14024oooooooooooos Pmff00fff00m0m UUUAGCUCGGUAUGUCU 110 16-14024 ssssso0mmmm0 UC CTGF-2264- 14025 oooooooooooos Pmf0fffff00m00ACACUCUCAACAAAUAA 111 16-14025 ssssso 0m0000 AC CTGF-1032- 14026oooooooooooos Pm00fff00f0m0m UAGCUCGGUAUGUCUUC 112 16-14026 sssssommmm00 AU CTGF-1535- 14027 oooooooooooos Pm00fffffff0mmUAACCUUUCUGCUGGUA 113 16-14027 ssssso 00m0m0 CC CTGF-1694- 14028oooooooooooos Pmf000000f00mm UUAAGGAACAACUUGAC 114 16-14028 sssssom00mm0 UC CTGF-1588- 14029 oooooooooooos Pmf0f0ffff000mUUACACUUCAAAUAGCA 115 16-14029 ssssso 00m000 GG CTGF-928- 14030oooooooooooos Pmff000ff00mmm UCCAGGUCAGCUUCGCA 116 16-14030 sssssom0m000 AG CTGF-1133- 14031 oooooooooooos Pmffffff0f00mmCUUCUUCAUGACCUCGC 117 16-14031 ssssso mm0mm0 CG CTGF-912- 14032oooooooooooos Pm000fff00fm0m AAGGCCUGACCAUGCAC 118 16-14032 ssssso0m0m00 AG CTGF-753- 14033 oooooooooooos Pm000f0f0ffmmm CAAACGUGUCUUCCAGU119 16-14033 ssssso m00mm0 CG CTGF-918- 14034 oooooooooooosPmfff0f0000mmm CUUCGCAAGGCCUGACC 120 16-14034 ssssso 00mm00 AU CTGF-744-14035 oooooooooooos Pmffff00ff00m0 CUUCCAGUCGGUAAGCC 121 16-14035 ssssso00mm00 GC CTGF-466- 14036 oooooooooooos Pmf00ffff0f00m CCGAUCUUGCGGUUGGC122 16-14036 ssssso m00mm0 CG CTGF-917- 14037 oooooooooooosPmff0f0000fmm0 UUCGCAAGGCCUGACCA 123 16-14037 ssssso 0mm0m0 UGCTGF-1038- 14038 oooooooooooos Pm00fff00fmm0m AGAAUUUAGCUCGGUAU 12416-14038 ssssso 0m00 GU CTGF-1048- 14039 oooooooooooos Pm0f0ffff0f000CAUACUCCACAGAAUUU 125 16-14039 ssssso 0mmm00 AG CTGF-1235- 14040oooooooooooos Pm0ff0f0fffmmm UGCCAUGUCUCCGUACA 126 16-14040 ssssso 0m0m0UC CTGF-868- 14041 oooooooooooos Pm000f0ff0fm0m GAGGCGUUGUCAUUGGU 12716-14041 ssssso m00m00 AA CTGF-1131- 14042 oooooooooooos Pmffff0f00fmmmUCUUCAUGACCUCGCCG 128 16-14042 ssssso 0mm0m0 UC CTGF-1043- 14043oooooooooooos Pmff0f0000fmm0 UCCACAGAAUUUAGCUC 129 16-14043 ssssso0mmm00 GG CTGF-751- 14044 oooooooooooos Pm0f0f0ffffmm0 AACGUGUCUUCCAGUCG130 16-14044 ssssso 0mm000 GU CTGF-1227- 14045 oooooooooooosPmfff0f0f0fmmm CUCCGUACAUCUUCCUG 131 16-14045 ssssso mmm0m0 UA CTGF-867-14046 oooooooooooos Pm0f0ff0ff0mm0 AGGCGUUGUCAUUGGUA 132 16-14046 ssssso0m000 AC CTGF-1128- 14047 oooooooooooos Pmf0f00ffff0mm UCAUGACCUCGCCGUCA133 16-14047 ssssso 0mm000 GG CTGF-756- 14048 oooooooooooosPm0ff000f0f0mm GGCCAAACGUGUCUUCC 134 16-14048 ssssso mmmm00 AGCTGF-1234- 14049 oooooooooooos Pmff0f0ffffmm0 GCCAUGUCUCCGUACAU 13516-14049 ssssso m0mm0 CU CTGF-916- 14050 oooooooooooos Pmf0f0000ffm00UCGCAAGGCCUGACCAU 16-14050 ssssso mm0m00 GC 136 CTGF-925- 14051oooooooooooos Pm0ff00fffmm00 AGGUCAGCUUCGCAAGG 137 16-14051 ssssso 00m0CC CTGF-1225- 14052 oooooooooooos Pmf0f0f0fffmmm CCGUACAUCUUCCUGUA 13816-14052 ssssso m0m000 GU CTGF-445- 14053 oooooooooooos Pm00ff0000fm0mGAGCCGAAGUCACAGAA 16-14053 ssssso 000000 GA 139 CTGF-446- 14054oooooooooooos Pm000ff0000mm0 GGAGCCGAAGUCACAGA 140 16-14054 sssssom00000 AG CTGF-913- 14055 oooooooooooos Pm0000fff00mm0 CAAGGCCUGACCAUGCA141 16-14055 ssssso m0m0m0 CA CTGF-997- 14056 oooooooooooosPmfff000ffm00m AGCUCAAACUUGAUAGG 142 16-14056 ssssso 000m0 CU CTGF-277-14057 oooooooooooos Pmf0f00ffff00m CUGCAGUUCUGGCCGAC 143 16-14057 sssssom00m00 GG CTGF-1052- 14058 oooooooooooos Pm0f0f0f0ffmm0GGUACAUACUCCACAGA 144 16-14058 ssssso m00000 AU CTGF-887- 14059oooooooooooos Pmf0fffffff00m CUGCUUCUCUAGCCUGC 145 16-14059 sssssomm0m00 AG CTGF-914- 14060 oooooooooooos Pmf0000fff00mm GCAAGGCCUGACCAUGC146 16-14060 ssssso 0m0m00 AC CTGF-1039- 14061 oooooooooooosPm0000fff00mmm CAGAAUUUAGCUCGGUA 147 16-14061 ssssso 00m0m0 UG CTGF-754-14062 oooooooooooos Pmf000f0f0fmmm CCAAACGUGUCUUCCAG 148 16-14062 sssssomm00m0 UC CTGF-1130- 14063 oooooooooooos Pmfff0f00ffmmmCUUCAUGACCUCGCCGU 149 16-14063 ssssso m0mm0 CA CTGF-919- 14064oooooooooooos Pmffff0f0000mm GCUUCGCAAGGCCUGAC 150 16-14064 sssssom00mm0 CA CTGF-922- 14065 oooooooooooos Pmf00ffff0f000 UCAGCUUCGCAAGGCCU151 16-14065 ssssso 0mmm00 GA CTGF-746- 14066 oooooooooooosPmffffff00fm0m GUCUUCCAGUCGGUAAG 152 16-14066 ssssso 000m0 CC CTGF-993-14067 oooooooooooos Pm000fff00f000 CAAACUUGAUAGGCUUG 153 16-14067 sssssommm000 GA CTGF-825- 14068 oooooooooooos Pm0ffff0000m00 AGGUCUUGGAACAGGCG154 16-14068 ssssso 0m0m0 CU CTGF-926- 14069 oooooooooooosPm000ff00ffmmm CAGGUCAGCUUCGCAAG 155 16-14069 ssssso 00000 GC CTGF-923-14070 oooooooooooos Pmff00ffff0m00 GUCAGCUUCGCAAGGCC 156 16-14070 ssssso00mmm0 UG CTGF-866- 14071 oooooooooooos Pm0f0ff0ff0mm0 GGCGUUGUCAUUGGUAA157 16-14071 ssssso 0m00m0 CC CTGF-563- 14072 oooooooooooosPmf0f0ff00m0mm CGUGCACUGGUACUUGC 158 16-14072 ssssso m0m00 AG CTGF-823-14073 oooooooooooos Pmffff0000f000 GUCUUGGAACAGGCGCU 159 16-14073 sssssom0mmm0 CC CTGF-1233- 14074 oooooooooooos Pmf0f0fffff0m0CCAUGUCUCCGUACAUC 160 16-14074 ssssso m0mmm0 UU CTGF-924- 14075oooooooooooos Pm0ff00ffff0m0 GGUCAGCUUCGCAAGGC 161 16-14075 ssssso000mm0 CU CTGF-921- 14076 oooooooooooos Pm00ffff0f0000 CAGCUUCGCAAGGCCUG162 16-14076 ssssso mmm000 AC CTGF-443- 14077 oooooooooooosPmff0000ff0m00 GCCGAAGUCACAGAAGA 163 16-14077 ssssso 000000 GGCTGF-1041- 14078 oooooooooooos Pm0f0000fff00m CACAGAAUUUAGCUCGG 16416-14078 ssssso mm00m0 UA CTGF-1042- 14079 oooooooooooos Pmf0f0000ffm00CCACAGAAUUUAGCUCG 165 16-14079 ssssso mmm000 GU CTGF-755- 14080oooooooooooos Pmff000f0f0mmm GCCAAACGUGUCUUCCA 166 16-14080 sssssommm000 GU CTGF-467- 14081 oooooooooooos Pmf0f00ffff0m0 GCCGAUCUUGCGGUUGG167 16-14081 ssssso mm00m0 CC CTGF-995- 14082 oooooooooooosPmff000fff00m0 CUCAAACUUGAUAGGCU 168 16-14082 ssssso 00mmm0 UG CTGF-927-14083 oooooooooooos Pmf000ff00fmmm CCAGGUCAGCUUCGCAA 169 16-14083 ssssso0m0000 GG SPP1-1091- 14131 oooooooooooos Pmff00ff000m0mUUUGACUAAAUGCAAAG 170 16-14131 ssssso 0000m0 UG PPIB--16- 14188ooooooooooooo Pm0fffff0f00mm UGUUUUUGUAGCCAAAU 171 14188 ssssss 000mm0CC PPIB--17- 14189 ooooooooooooo Pm0fffff0f00mm UGUUUUUGUAGCCAAAU 17214189 ssssss 000mm0 CC PPIB--18- 14190 ooooooooooooo Pm0fffff0f00mmUGUUUUUGUAGCCAAAU 173 14190 ssssss 000mm0 CC pGL3-1172- 14386oooooooooooos Pm00ff0f0ffm0m AAAUCGUAUUUGUCAAU 174 16-14386 sssssom00mm0 CA pGL3-1172- 14387 oooooooooooos Pm00ff0f0ffm0mAAAUCGUAUUUGUCAAU 175 16-14387 ssssso m00mm0 CA MAP4K4- 14390 2931-25-14390 miR-122-- 14391 23-14391 14084 oooooooooooos Pmff00fff0f000UCUAAUUCAUGAGAAAU 616 ssssso 000m00 AC 14085 oooooooooooosPm00ff00fffm00 UAAUUGACCUCAGAAGA 617 ssssso 0000m0 UG 14086oooooooooooos Pmff00ff00fmmm UUUAAUUGACCUCAGAA 618 ssssso 000000 GA14087 oooooooooooos Pm0ff00ffff000 AAUUGACCUCAGAAGAU 619 ssssso 000m00GC 14088 oooooooooooos Pmf00ff00ffmm0 UUAAUUGACCUCAGAAG 620 ssssso000000 AU 14089 oooooooooooos Pmff00ffff0000 AUUGACCUCAGAAGAUG 621ssssso 00m0m0 CA 14090 oooooooooooos Pmf0fff00ff00m UCAUCCAGCUGACUCGU622 ssssso mm0mm0 UU 14091 oooooooooooos Pm0fff0ff0000mAGAUUCAUCAGAAUGGU 623 ssssso 00m00 GA 14092 oooooooooooos Pm00ffff00fmm0UGACCUCAGUCCAUAAA 624 ssssso m000m0 CC 14093 oooooooooooosPm0f00f0000mmm AAUGGUGAGACUCAUCA 625 ssssso 0mm000 GA 14094oooooooooooos Pmff00ffff00mm UUUGACCUCAGUCCAUA 626 ssssso m0m000 AA14095 oooooooooooos Pmff0f00ff0m00 UUCAUGGCUGUGAAAUU 627 ssssso 00mmm0CA 14096 oooooooooooos Pm00f00f0000mm GAAUGGUGAGACUCAUC 628 sssssom0mm00 AG 14097 oooooooooooos Pm00ffffff0mmm UGGCUUUCCGCUUAUAU 629ssssso 0m0m00 AA 14098 oooooooooooos Pmf00ffffff0mm UUGGCUUUCCGCUUAUA630 ssssso m0m0m0 UA 14099 oooooooooooos Pmf0fff0f0f00mUCAUCCAUGUGGUCAUG 631 ssssso m0m000 GC 14100 oooooooooooosPmf0f00ff0f00m AUGUGGUCAUGGCUUUC 632 ssssso mmmm00 GU 14101oooooooooooos Pmf00ff0f00mmm GUGGUCAUGGCUUUCGU 633 ssssso mm0mm0 UG14102 oooooooooooos Pmff00fffffmmm AUUGGCUUUCCGCUUAU 634 ssssso m0m00 AU14103 oooooooooooos Pm00f0f0000mmm AAAUACGAAAUUUCAGG 635 ssssso m000m0UG 14104 oooooooooooos Pm000f0f0000mm AGAAAUACGAAAUUUCA 636 ssssso mm000GG 14105 oooooooooooos Pm00ff0f00fmmm UGGUCAUGGCUUUCGUU 637 sssssom0mm00 GG 14106 oooooooooooos Pmf0ff0fff0m0m AUAUCAUCCAUGUGGUC 638ssssso 00mm00 AU 14107 oooooooooooos Pm0f0f0000fmmm AAUACGAAAUUUCAGGU639 ssssso 000m00 GU 14108 oooooooooooos Pm0ff000000mm0AAUCAGAAGGCGCGUUC 640 ssssso mmm00 AG 14109 oooooooooooos Pmfff0f000000mAUUCAUGAGAAAUACGA 641 ssssso 0m0000 AA 14110 oooooooooooosPmf0fff0f00000 CUAUUCAUGAGAGAAUA 642 ssssso 00m000 AC 14111oooooooooooos Pmfff0ff000mmm UUUCGUUGGACUUACUU 643 ssssso 0mmm00 GG14112 oooooooooooos Pmf0fffff0fm0m UUGCUCUCAUCAUUGGC 644 ssssso m00mm0UU 14113 oooooooooooos Pmff00fffffmmm UUCAACUCCUCGCUUUC 645 ssssso mmmm0CA 14114 oooooooooooos Pm00ff0ff00mm0 UGACUAUCAAUCACAUC 646 sssssom0mm00 GG 14115 oooooooooooos Pm0f0f0ff0mmm0 AGAUGCACUAUCUAAUU 647ssssso 0mmm0 CA 14116 oooooooooooos Pm0f000f0f0m0m AAUAGAUACACAUUCAA 648ssssso mm00m0 CC 14117 oooooooooooos Pmffffff0f0000 UUCUUCUAUAGAAUGAA649 ssssso m000m0 CA 14118 oooooooooooos Pm0ff0ff000m00AAUUGCUGGACAACCGU 650 ssssso mm0m00 GG 14119 oooooooooooosPmf0ffffff0m0m UCGCUUUCCAUGUGUGA 651 ssssso 0m0000 GG 14120oooooooooooos Pm00fff000fm0m UAAUCUGGACUGCUUGU 652 ssssso mm0m00 GG14121 oooooooooooos Pmf0f0fff00mm0 ACACAUUCAACCAAUAA 653 ssssso 0m0000AC 14122 oooooooooooos Pmfff0ffff0m00 ACUCGUUUCAUAACUGU 654 sssssomm0mm0 CC 14123 oooooooooooos Pmf00fff000mm0 AUAAUCUGGACUGCUUG 655ssssso mmm0m0 UG 14124 oooooooooooos Pmffff0fff0m0m UUUCCGCUUAUAUAAUC656 ssssso 00mmm0 UG 14125 oooooooooooos Pm0fff00ff00m0UGUUUAACUGGUAUGGC 657 ssssso m00m00 AC 14126 oooooooooooosPm0f0000f000m0 UAUAGAAUGAACAUAGA 658 ssssso m000m0 CA 14127oooooooooooos Pmffffff00fm0m UUUCCUUGGUCGGCGUU 659 ssssso 0mmm0 UG 14128oooooooooooos Pmf0f0f0ff0mmm GUAUGCACCAUUCAACU 660 ssssso 00mmm0 CC14129 oooooooooooos Pmf00ff0ff0m0m UCGGCCAUCAUAUGUGU 661 ssssso 0m0mm0CU 14130 oooooooooooos Pm0fff000ff0mm AAUCUGGACUGCUUGUG 662 sssssom0m000 GC 14132 oooooooooooos Pmf0ff0000f0mm ACAUCGGAAUGCUCAUU 663ssssso m0mm00 GC 14133 oooooooooooos Pm00fffff00mm0 AAGUUCCUGACUAUCAA664 ssssso mm00m0 UC 14134 oooooooooooos Pmf00ff000f0m0UUGACUAAAUGCAAAGU 665 ssssso 000m00 GA 14135 oooooooooooosPm0fff0ff000mm AGACUCAUCAGACUGGU 666 ssssso 00m00 GA 14136 oooooooooooosPmf0f0f0f0fmm0 UCAUAUGUGUCUACUGU 667 ssssso mm0m00 GG 14137oooooooooooos Pmf0fffff0fmm0 AUGUCCUCGUCUGUAGC 668 ssssso m00m00 AU14138 oooooooooooos Pm00fff0f00mm0 GAAUUCACGGCUGACUU 669 ssssso 0mmmm0UG 14139 oooooooooooos Pmf0fffff000mm UUAUUUCCAGACUCAAA 670 sssssom000m0 UA 14140 oooooooooooos Pm000ff0f000mm GAAGCCACAAACUAAAC 671ssssso 000mm0 UA 14141 oooooooooooos Pmffff0ff000mm CUUUCGUUGGACUUACU672 ssssso m0mmm0 UG 14142 oooooooooooos Pmfff0f0000mmmGUCUGCGAAACUUCUUA 673 ssssso mmm000 GA 14143 oooooooooooosPm0f0fff0ff0mm AAUGCUCAUUGCUCUCA 674 ssssso mmm0m0 UC 14144oooooooooooos Pmf0f0ff0ffm00 AUGCACUAUCUAAUUCA 675 ssssso mmm0m0 UG14145 oooooooooooos Pmff0f0f0f0mm0 CUUGUAUGCACCAUUCA 676 ssssso mmm000AC 14146 oooooooooooos Pm00fff0fffm0m UGACUCGUUUCAUAACU 677 ssssso00mm00 GU 14147 oooooooooooos Pmff00f0fffm00 UUCAGCACUCUGGUCAU 678ssssso mm0mm0 CC 14148 oooooooooooos Pm00fff0f00mm0 AAAUUCAUGGCUGUGGA679 ssssso m00000 AU 14149 oooooooooooos Pmf0fff00ff00mACAUUCAACCAAUAAAC 680 ssssso 000mm0 UG 14150 oooooooooooosPm0f0f0fff00mm UACACAUUCAACCAAUA 681 ssssso 00m000 AA 14151oooooooooooos Pmff00ff0ffmmm AUUAGUUAUUUCCAGAC 682 ssssso 000mm0 UC14152 oooooooooooos Pmffff0fff0m00 UUUCUAUUCAUGAGAGA 683 ssssso 000000AU 14153 oooooooooooos Pmff00ff0ff00m UUCGGUUGCUGGCAGGU 684 ssssso000mm0 CC 14154 oooooooooooos Pm0f0f0f0000m0 CAUGUGUGAGGUGAUGU 685ssssso 0m0mm0 CC 14155 oooooooooooos Pmf0ff0fff00mm GCACCAUUCAACUCCUC686 ssssso mmmm00 GC 14156 oooooooooooos Pm0fff00ff00mmCAUCCAGCUGACUCGUU 687 ssssso m0mmm0 UC 14157 oooooooooooosPmfffff0fff0m0 CUUUCCGCUUAUAUAAU 688 ssssso m00mm0 CU 14158oooooooooooos Pm0ff0f0ff0000 AAUCACAUCGGAAUGCU 689 ssssso m0mmm0 CA14159 oooooooooooos Pmf0f0ff00fm0m ACACAUUAGUUAUUUCC 690 ssssso mmmm00AG 14160 oooooooooooos Pmfff0f0000m00 UUCUAUAGAAUGAACAU 691 ssssso0m0m00 AG 14161 oooooooooooos Pm0f00f00f00mm UACAGUGAUAGUUUGCA 692ssssso m0m0m0 UU 14162 oooooooooooos Pmf000f00ff00m AUAAGCAAUUGACACCA693 ssssso 0mm0m0 CC 14163 oooooooooooos Pmff0ff00ff0mmUUUAUUAAUUGCUGGAC 694 ssssso 000m00 AA 14164 oooooooooooosPmf0ff0000fmmm UCAUCAGAGUCGUUCGA 695 ssssso m0000 GU 14165 oooooooooooosPmf000ff0f0mm0 AUAAACCACACUAUCAC 696 ssssso mm0mm0 CU 14166oooooooooooos Pmf0ff0ff00mmm UCAUCAUUGGCUUUCCG 697 ssssso mmm0m0 CU14167 oooooooooooos Pmfffff00fm0mm AGUUCCUGACUAUCAAU 698 ssssso 00mm0 CA14168 oooooooooooos Pmff0f00ff00mm UUCACGGCUGACUUUGG 699 ssssso mm0000AA 14169 oooooooooooos Pmffff0f00f00m UUCUCAUGGUAGUGAGU 700 ssssso000mm0 UU 14170 oooooooooooos Pm0ff00fff0mmm AAUCAGCCUGUUUAACU 701ssssso 00mm00 GG 14171 oooooooooooos Pm0ffff00f0mmm GGUUUCAGCACUCUGGU702 ssssso m00mm0 CA 14172 oooooooooooos Pmff0000f0fmm0AUCGGAAUGCUCAUUGC 703 ssssso mm0mm0 UC 14173 oooooooooooosPm00ff0f0000mm UGGCUGUGGAAUUCACG 704 ssssso m0m000 GC 14174oooooooooooos Pm000f00ff00m0 UAAGCAAUUGACACCAC 705 ssssso mm0mm0 CA14175 oooooooooooos Pm00fffff0f00m CAAUUCUCAUGGUAGUG 706 ssssso 00m000AG 14176 oooooooooooos Pm00fffff0fm00 UGGCUUUCGUUGGACUU 707 ssssso0mmm00 AC 14177 oooooooooooos Pm0ff00f00fm00 AAUCAGUGACCAGUUCA 708ssssso mmm0m0 UC 14178 oooooooooooos Pmfff0f000mm0m AGUCCAUAAACCACACU709 ssssso 0mm00 AU 14179 oooooooooooos Pm00f0ffff00mm CAGCACUCUGGUCAUCC710 ssssso 0mmm00 AG 14180 oooooooooooos Pm0ff00ff0f0mmUAUCAAUCACAUCGGAA 711 ssssso 0000m0 UG 14181 oooooooooooosPmfff0f00ff00m AUUCACGGCUGACUUUG 712 ssssso mmm000 GA 14182oooooooooooos Pmf000f0f0f0mm AUAGAUACACAUUCAAC 713 ssssso m00mm0 CA14183 oooooooooooos Pmffff000ffm00 UUUCCAGACUCAAAUAG 714 ssssso 0m0000AU 14184 oooooooooooos Pmf00ff0ff000m UUAAUUGCUGGACAACC 715 ssssso00mm00 GU 14185 oooooooooooos Pm0ff00ff0fm00 UAUUAAUUGCUGGACAA 716ssssso 0m00m0 CC 14186 oooooooooooos Pmff0fff000mm0 AGUCGUUCGAGUCAAUG717 ssssso 0m000 GA 14187 oooooooooooos Pmff0ff00f000m GUUGCUGGCAGGUCCGU718 ssssso mm0m00 GG

TABLE 3Sense backbone, chemistry, and sequence information. o: phosphodiester; s:phosphorothioate; P: 5′phosphorylation; 0: 2′-OH; F: 2′-fluoro; m: 2′O-methyl;+: LNA modification. Capital letters in the sequence signify ribonucleotides,lower case letters signify deoxyribonucleotides. OHang SEQ Oligo SenseSense Sense Sense ID ID Number Number Chem. Backbone Chemistry SequenceNO: APOB-10167- 12138 chl oooooooooooo 0000000000000 GUCAUCACACUGA 17620-12138 oooooooso 0000000 AUACCAAU APOB-10167- 12139 chl oooooooooooo0000000000000 GUGAUCAGACUCA 177 20-12139 oooooooso 0000000 AUACGAAUMAP4K4- 12266 chl ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 178 2931-13-o 12266 MAP4K4- 12293 chl ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 1792931-16- o 12293 MAP4K4- 12383 chl oooooooooooo mm0m00000mmm0CUGUGGAAGUCUA 180 2931-16- o 12383 MAP4K4- 12384 chl oooooooooooomm0m00000mmm0 CUGUGGAAGUCUA 181 2931-16- o 12384 MAP4K4- 12385 chloooooooooooo mm0m00000mmm0 CUGUGGAAGUCUA 182 2931-16- o 12385 MAP4K4-12386 chl ooooooooooss 0mm0m00000mmm CUGUGGAAGUCUA 183 2931-16- o 012386 MAP4K4- 12387 chl oooooooooooo mm0m00000mmm0 CUGUGGAAGUCUA 1842931-16- o 12387 MAP4K4- 12388 chl oooooooooooo mm0m00000mmm0CUGUGGAAGUCUA 185 2931-15- o 12388 MAP4K4- 12432 chl ooooooooooooDY547mm0m0000 CUGUGGAAGUCUA 186 2931-13- o 0mmm0 12432 MAP4K4- 12266.2chl ooooooooooos mm0m00000mmm0 CUGUGGAAGUCUA 187 2931-13- s 12266.2APOB--21- 12434 chl oooooooooooo 0000000000000 GUCAUCACACUGA 188 12434oooooooso 0000000 AUACCAAU APOB--21- 12435 chl ooooooooooooDY54700000000 GUGAUCAGACUCA 189 12435 oooooooso 000000000000 AUACGAAUMAP4K4- 12451 chl ooooooooooos 0mm0m00000mmm CUGUGGAAGUCUA 190 2931-16-s 0 12451 MAP4K4- 12452 chl ooooooooooos mm0m00000mmm0 CUGUGGAAGUCUA 1912931-16- s 12452 MAP4K4- 12453 chl ooooooooooos mm0m00000mmm0CUGUGGAAGUCUA 192 2931-16- s 12453 MAP4K4- 12454 chl ooooooooooos0mm0m00000mmm CUGUGGAAGUCUA 193 2931-17- s 0 12454 MAP4K4- 12455 chlooooooooooos mm0m00000mmm0 CUGUGGAAGUCUA 194 2931-17- s 12455 MAP4K4-12456 chl ooooooooooos mm0m00000mmm0 CUGUGGAAGUCUA 195 2931-19- s 12456--27-12480 12480 chl oooooooooooo DY547mm0f000f UCAUAGGUAACCU 196oooooooooooo 0055f5f00mm00 CUGGUUGAAAGUG sso 000m000 A --27-12481 12481chl oooooooooooo DY547mm05f050 CGGCUACAGGUGC 197 oooooooooooo00f05ff0m0000 UUAUGAAGAAAGU sso 0000m00 A APOB-10167- 12505 chloooooooooooo 0000000000000 GUCAUCACACUGA 198 21-12505 oooooooos 00000000AUACCAAU APOB-10167- 12506 chl oooooooooooo 0000000000000 GUGAUCAGACUCA199 21-12506 oooooooos 00000000 AUACGAAU MAP4K4- 12539 chl ooooooooooosDY547mm0m0000 CUGUGGAAGUCUA 200 2931-16- s 0mmm0 12539 APOB-10167-12505.2 chl oooooooooooo 0000000000000 GUCAUCACACUGA 201 21-12505.2oooooooso 0000000 AUACCAAU APOB-10167- 12506.2 chl oooooooooooo0000000000000 GUGAUCAGACUCA 202 21-12506.2 oooooooso 0000000 AUACGAAUMAP4K4--13- 12565 Chl oooooooooooo m0m0000m0mmm0 UGUAGGAUGUCUA 203 12565o MAP4K4- 12386.2 chl oooooooooooo 0mm0m00000mmm CUGUGGAAGUCUA 2042931-16- o 0 12386.2 MAP4K4- 12815 chl oooooooooooo m0m0m0m0m0m0mCUGUGGAAGUCUA 205 2931-13- o 0m0m0m0m0m0m0 12815 APOB--13- 12957 Chlooooooooooos 0mmmmmmmmmmmm ACUGAAUACCAAU 206 12957 TEG s m MAP4K4--16-12983 chl ooooooooooos mm0m00000mmm0 CUGUGGAAGUCUA 207 12983 sMAP4K4--16- 12984 Chl oooooooooooo mm0m00000mmm0 CUGUGGAAGUCUA 208 12984oo MAP4K4--16- 12985 chl ooooooooooss mmmmmmmmmmmmm CUGUGGAAGUCUA 20912985 o MAP4K4--16- 12986 chl ooooooooooss mmmmmmmmmmmmm CUGUGGAAGUCUA210 12986 o MAP4K4--16- 12987 chl ooooooooooss mm0m00000mmm0CUGUGGAAGUCUA 211 12987 o MAP4K4--16- 12988 chl oooooooooossmm0m00000mmm0 CUGUGGAAGUCUA 212 12988 o MAP4K4--16- 12989 chlooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 213 12989 o MAP4K4--16- 12990chl ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 214 12990 o MAP4K4--16-12991 chl ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 215 12991 oMAP4K4--16- 12992 chl ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 216 12992o MAP4K4--16- 12993 chl ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 21712993 o MAP4K4--16- 12994 chl ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA218 12994 o MAP4K4--16- 12995 chl ooooooooooss mm0m00000mmm0CUGUGGAAGUCUA 219 12995 o MAP4K4- 13012 chl oooooooooooo 0000000000000AGAGUUCUGUGGA 220 2931-19- ooooooo 00000000 AGUCUA 13012 MAP4K4- 13016chl oooooooooooo DY54700000000 AGAGUUCUGUGGA 221 2931-19- ooooooo0000000000000 AGUCUA 13016 PPIB--13- 13021 Chl oooooooooooo0mmm00mm0m000 AUUUGGCUACAAA 222 13021 o pGL3-1172- 13038 chloooooooooooo 00m000m0m00mm ACAAAUACGAUUU 223 13-13038 o m pGL3-1172-13040 chl oooooooooooo DY5470m000m0m ACAAAUACGAUUU 224 13-13040 o 00mmm--16-13047 13047 Chl oooooooooooo mm0m00000mmm0 CUGUGGAAGUCUA 225 ooSOD1-530- 13090 chl oooooooooooo 00m00000000m0 AAUGAAGAAAGUA 22613-13090 o SOD1-523- 13091 chl oooooooooooo 000m00000m000 AGGUGGAAAUGAA227 13-13091 o SOD1-535- 13092 chl oooooooooooo 000000m0m0000AGAAAGUACAAAG 228 13-13092 o SOD1-536- 13093 chl oooooooooooo00000m0m00000 GAAAGUACAAAGA 229 13-13093 o SOD1-396- 13094 chloooooooooooo 0m0m00mm0mm00 AUGUGACUGCUGA 230 13-13094 o SOD1-385- 13095chl oooooooooooo 000mmm000m00m AGACUUGGGCAAU 231 13-13095 o SOD1-195-13096 chl oooooooooooo 0mmmm000m0000 AUUUCGAGCAGAA 232 13-13096 oAPOB-4314- 13115 Chl oooooooooooo 0mmm0000000m0 AUCUGGAGAAACA 23313-13115 o APOB-3384- 13116 Chl oooooooooooo mm0000m000000 UCAGAACAAGAAA234 13-13116 o APOB-3547- 13117 Chl oooooooooooo 00mmm0mmm0mm0GACUCAUCUGCUA 235 13-13117 o APOB-4318- 13118 Chl oooooooooooo0000000m00m0m GGAGAAACAACAU 236 13-13118 o APOB-3741- 13119 Chloooooooooooo 00mmmmmm000m0 AGUCCCUCAAACA 237 13-13119 o PPIB--16- 13136Chl oooooooooooo 00mm0m00000m0 GGCUACAAAAACA 238 13136 oo APOB-4314-13154 chl oooooooooooo 000mmm0000000 AGAUCUGGAGAAA 239 15-13154 oo m0 CAAPOB-3547- 13155 chl oooooooooooo m000mmm0mmm0m UGGACUCAUCUGC 24015-13155 oo m0 UA APOB-4318- 13157 chl oooooooooooo mm0000000m00mCUGGAGAAACAAC 241 15-13157 oo 0m AU APOB-3741- 13158 chl oooooooooooo0000mmmmmm000 AGAGUCCCUCAAA 242 15-13158 oo m0 CA APOB--13- 13159 chloooooooooooo 0mm000mOmm00m ACUGAAUACCAAU 243 13159 APOB--15- 13160 chloooooooooooo 0m0mm000m0mm0 ACACUGAAUACCA 244 13160 oo 0m AU SOD1-530-13163 chl oooooooooooo 00m00000000m0 AAUGAAGAAAGUA 245 16-13163 oSOD1-523- 13164 chl oooooooooooo 000m00000m000 AGGUGGAAAUGAA 24616-13164 o SOD1-535- 13165 chl oooooooooooo 000000m0m0000 AGAAAGUACAAAG247 16-13165 o SOD1-536- 13166 chl oooooooooooo 00000m0m00000GAAAGUACAAAGA 248 16-13166 o SOD1-396- 13167 chl oooooooooooo0m0m00mm0mm00 AUGUGACUGCUGA 249 16-13167 o SOD1-385- 13168 chloooooooooooo 000mmm000m00m AGACUUGGGCAAU 250 16-13168 o SOD1-195- 13169chl oooooooooooo 0mmmm000m0000 AUUUCGAGCAGAA 251 16-13169 o pGL3-1172-13170 chl oooooooooooo 0m000m0m00mmm ACAAAUACGAUUU 252 16-13170 opGL3-1172- 13171 chl oooooooooooo DY5470m000m0m ACAAAUACGAUUU 25316-13171 o 00mmm MAP4k4- 13189 chl oooooooooooo 0000000000000AGAGUUCUGUGGA 254 2931-19- ooooooo 00000000 AGUCUA 13189 CTGF-1222-13190 Chl oooooooooooo 0m0000000m0m0 ACAGGAAGAUGUA 255 13-13190 oCTGF-813- 13192 Chl oooooooooooo 000m0000m0mmm GAGUGGAGCGCCU 25613-13192 o CTGF-747- 13194 Chl oooooooooooo m00mm000000m0 CGACUGGAAGACA257 13-13194 o CTGF-817- 13196 Chl oooooooooooo 0000m0mmm0mmmGGAGCGCCUGUUC 258 13-13196 o CTGF-1174- 13198 Chl oooooooooooo0mm0mm0m00mm0 GCCAUUACAACUG 259 13-13198 o CTGF-1005- 13200 Chloooooooooooo 000mmmmmm00mm GAGCUUUCUGGCU 260 13-13200 o CTGF-814- 13202Chl oooooooooooo 00m0000m0mmm0 AGUGGAGCGCCUG 261 13-13202 o CTGF-816-13204 Chl oooooooooooo m0000m0mmm0mm UGGAGCGCCUGUU 262 13-13204 oCTGF-1001- 13206 Chl oooooooooooo 0mmm000mmmmmm GUUUGAGCUUUCU 26313-13206 o CTGF-1173- 13208 Chl oooooooooooo m0mm0mm0m00mm UGCCAUUACAACU264 13-13208 o CTGF-749- 13210 Chl oooooooooooo 0mm000000m0m0ACUGGAAGACACG 265 13-13210 o CTGF-792- 13212 Chl oooooooooooo00mm0mmm00mmm AACUGCCUGGUCC 266 13-13212 o CTGF-1162- 13214 Chloooooooooooo 000mmm0m0mmm0 AGACCUGUGCCUG 267 13-13214 o CTGF-811- 13216Chl oooooooooooo m0000m0000m0m CAGAGUGGAGCGC 268 13-13216 o CTGF-797-13218 Chl oooooooooooo mmm00mmm000mm CCUGGUCCAGACC 269 13-13218 oCTGF-1175- 13220 Chl oooooooooooo mm0mm0m00mm0m CCAUUACAACUGU 27013-13220 o CTGF-1172- 13222 Chl oooooooooooo mm0mm0mm0m00m CUGCCAUUACAAC271 13-13222 o CTGF-1177- 13224 Chl oooooooooooo 0mm0m00mm0mmmAUUACAACUGUCC 272 13-13224 o CTGF-1176- 13226 Chl oooooooooooom0mm0m00mm0mm CAUUACAACUGUC 273 13-13226 o CTGF-812- 13228 Chloooooooooooo 0000m0000m0mm AGAGUGGAGCGCC 274 13-13228 o CTGF-745- 13230Chl oooooooooooo 0mm00mm000000 ACCGACUGGAAGA 275 13-13230 o CTGF-1230-13232 Chl oooooooooooo 0m0m0m00000m0 AUGUACGGAGACA 276 13-13232 oCTGF-920- 13234 Chl oooooooooooo 0mmmm0m0000mm GCCUUGCGAAGCU 27713-13234 o CTGF-679- 13236 Chl oooooooooooo 0mm0m000000m0 GCUGCGAGGAGUG278 13-13236 o CTGF-992- 13238 Chl oooooooooooo 0mmm0mm000mmmGCCUAUCAAGUUU 279 13-13238 o CTGF-1045- 13240 Chl oooooooooooo00mmmm0m0000m AAUUCUGUGGAGU 280 13-13240 o CTGF-1231- 13242 Chloooooooooooo m0m0m00000m0m UGUACGGAGACAU 281 13-13242 o CTGF-991- 13244Chl oooooooooooo 00mmm0mm000mm AGCCUAUCAAGUU 282 13-13244 o CTGF-998-13246 Chl oooooooooooo m000mmm000mmm CAAGUUUGAGCUU 283 13-13246 oCTGF-1049- 13248 Chl oooooooooooo mm0m0000m0m0m CUGUGGAGUAUGU 28413-13248 o CTGF-1044- 13250 Chl oooooooooooo 000mmmm0m0000 AAAUUCUGUGGAG285 13-13250 o CTGF-1327- 13252 Chl oooooooooooo mmmm00m00m0m0UUUCAGUAGCACA 286 13-13252 o CTGF-1196- 13254 Chl oooooooooooom00m00m0mmmmm CAAUGACAUCUUU 287 13-13254 o CTGF-562- 13256 Chloooooooooooo 00m0mm00m0m0m AGUACCAGUGCAC 288 13-13256 o CTGF-752- 13258Chl oooooooooooo 000000m0m0mmm GGAAGACACGUUU 289 13-13258 o CTGF-994-13260 Chl oooooooooooo mm0mm000mmm00 CUAUCAAGUUUGA 290 13-13260 oCTGF-1040- 13262 Chl oooooooooooo 00mm000mmmm0m AGCUAAAUUCUGU 29113-13262 o CTGF-1984- 13264 Chl oooooooooooo 000m0000m0m00 AGGUAGAAUGUAA292 13-13264 o CTGF-2195- 13266 Chl oooooooooooo 00mm00mm00mmmAGCUGAUCAGUUU 293 13-13266 o CTGF-2043- 13268 Chl oooooooooooommmm0mmm000m0 UUCUGCUCAGAUA 294 13-13268 o CTGF-1892- 13270 Chloooooooooooo mm0mmm000mm00 UUAUCUAAGUUAA 295 13-13270 o CTGF-1567- 13272Chl oooooooooooo m0m0m000m00m0 UAUACGAGUAAUA 296 13-13272 o CTGF-1780-13274 Chl oooooooooooo 00mm000m00mmm GACUGGACAGCUU 297 13-13274 oCTGF-2162- 13276 Chl oooooooooooo 0m00mmmmm0mm0 AUGGCCUUUAUUA 29813-13276 o CTGF-1034- 13278 Chl oooooooooooo 0m0mm000mm000 AUACCGAGCUAAA299 13-13278 o CTGF-2264- 13280 Chl oooooooooooo mm0mm00000m0mUUGUUGAGAGUGU 300 13-13280 o CTGF-1032- 13282 Chl oooooooooooo0m0m0mm000mm0 ACAUACCGAGCUA 301 13-13282 o CTGF-1535- 13284 Chloooooooooooo 00m0000000mm0 AGCAGAAAGGUUA 302 13-13284 o CTGF-1694- 13286Chl oooooooooooo 00mm0mmmmmm00 AGUUGUUCCUUAA 303 13-13286 o CTGF-1588-13288 Chl oooooooooooo 0mmm0000m0m00 AUUUGAAGUGUAA 304 13-13288 oCTGF-928- 13290 Chl oooooooooooo 000mm00mmm000 AAGCUGACCUGGA 30513-13290 o CTGF-1133- 13292 Chl oooooooooooo 00mm0m0000000 GGUCAUGAAGAAG306 13-13292 o CTGF-912- 13294 Chl oooooooooooo 0m00mm000mmmmAUGGUCAGGCCUU 307 13-13294 o CTGF-753- 13296 Chl oooooooooooo00000m0m0mmm0 GAAGACACGUUUG 308 13-13296 o CTGF-918- 13298 Chloooooooooooo 000mmmm0m0000 AGGCCUUGCGAAG 309 13-13298 o CTGF-744- 13300Chl oooooooooooo m0mm0mm00000 UACCGACUGGAAG 310 13-13300 o CTGF-466-13302 Chl oooooooooooo 0mm0m0000mm0 ACCGCAAGAUCGG 311 13-13302 oCTGF-917- 13304 Chl oooooooooooo m000mmmm0m000 CAGGCCUUGCGAA 31213-13304 o CTGF-1038- 13306 Chl oooooooooooo m000mm000mmmm CGAGCUAAAUUCU313 13-13306 o CTGF-1048- 13308 Chl oooooooooooo mmm0m0000m0m0UCUGUGGAGUAUG 314 13-13308 o CTGF-1235- 13310 Chl oooooooooooom00000m0m00m0 CGGAGACAUGGCA 315 13-13310 o CTGF-868- 13312 Chloooooooooooo 0m00m00m0mmmm AUGACAACGCCUC 316 13-13312 o CTGF-1131- 13314Chl oooooooooooo 0000mm0m00000 GAGGUCAUGAAGA 317 13-13314 o CTGF-1043-13316 Chl oooooooooooo m000mmmm0m000 UAAAUUCUGUGGA 318 13-13316 oCTGF-751- 13318 Chl oooooooooooo m000000m0m0mm UGGAAGACACGUU 31913-13318 o CTGF-1227- 13320 Chl oooooooooooo 0000m0m0m0000 AAGAUGUACGGAG320 13-13320 o CTGF-867- 13322 Chl oooooooooooo 00m00m00m0mmmAAUGACAACGCCU 321 13-13322 o CTGF-1128- 13324 Chl oooooooooooo00m0000mm0m00 GGCGAGGUCAUGA 322 13-13324 o CTGF-756- 13326 Chloooooooooooo 00m0m0mmm00mm GACACGUUUGGCC 323 13-13326 o CTGF-1234- 13328Chl oooooooooooo 0m00000m0m00m ACGGAGACAUGGC 324 13-13328 o CTGF-916-13330 Chl oooooooooooo mm000mmmm0m00 UCAGGCCUUGCGA 325 13-13330 oCTGF-925- 13332 Chl oooooooooooo 0m0000mm00mmm GCGAAGCUGACCU 32613-13332 o CTGF-1225- 13334 Chl oooooooooooo 000000m0m0m00 GGAAGAUGUACGG327 13-13334 o CTGF-445- 13336 Chl oooooooooooo 0m00mmmm00mmmGUGACUUCGGCUC 328 13-13336 o CTGF-446- 13338 Chl oooooooooooom00mmmm00mmmm UGACUUCGGCUCC 329 13-13338 o CTGF-913- 13340 Chloooooooooooo m00mm000mmmm0 UGGUCAGGCCUUG 330 13-13340 o CTGF-997- 13342Chl oooooooooooo mm000mmm000mm UCAAGUUUGAGCU 331 13-13342 o CTGF-277-13344 Chl oooooooooooo 0mm0000mm0m00 GCCAGAACUGCAG 332 13-13344 oCTGF-1052- 13346 Chl oooooooooooo m0000m0m0m0mm UGGAGUAUGUACC 33313-13346 o CTGF-887- 13348 Chl oooooooooooo 0mm0000000m00 GCUAGAGAAGCAG334 13-13348 o CTGF-914- 13350 Chl oooooooooooo 00mm000mmmm0mGGUCAGGCCUUGC 335 13-13350 o CTGF-1039- 13352 Chl oooooooooooo000mm000mmmm0 GAGCUAAAUUCUG 336 13-13352 o CTGF-754- 13354 Chloooooooooooo 0000m0m0mmm00 AAGACACGUUUGG 337 13-13354 o CTGF-1130- 13356Chl oooooooooooo m0000mm0m0000 CGAGGUCAUGAAG 338 13-13356 o CTGF-919-13358 Chl oooooooooooo 00mmmm0m0000m GGCCUUGCGAAGC 339 13-13358 oCTGF-922- 13360 Chl oooooooooooo mmm0m0000mm00 CUUGCGAAGCUGA 34013-13360 o CTGF-746- 13362 Chl oooooooooooo mm00mm000000m CCGACUGGAAGAC341 13-13362 o CTGF-993- 13364 Chl oooooooooooo mmm0mm000mmm0CCUAUCAAGUUUG 342 13-13364 o CTGF-825- 13366 Chl oooooooooooom0mmmm0000mmm UGUUCCAAGACCU 343 13-13366 o CTGF-926- 13368 Chloooooooooooo m0000mm00mmm0 CGAAGCUGACCUG 344 13-13368 o CTGF-923- 13370Chl oooooooooooo mm0m0000mm00m UUGCGAAGCUGAC 345 13-13370 o CTGF-866-13372 Chl oooooooooooo m00m00m00m0mm CAAUGACAACGCC 346 13-13372 oCTGF-563- 13374 Chl oooooooooooo 0m0mm00m0m0m0 GUACCAGUGCACG 34713-13374 o CTGF-823- 13376 Chl oooooooooooo mmm0mmmm0000m CCUGUUCCAAGAC348 13-13376 o CTGF-1233- 13378 Chl oooooooooooo m0m00000m0m00UACGGAGACAUGG 349 13-13378 o CTGF-924- 13380 Chl oooooooooooom0m0000mm00mm UGCGAAGCUGACC 350 13-13380 o CTGF-921- 13382 Chloooooooooooo mmmm0m0000mm0 CCUUGCGAAGCUG 351 13-13382 o CTGF-443- 13384Chl oooooooooooo mm0m00mmmm00m CUGUGACUUCGGC 352 13-13384 o CTGF-1041-13386 Chl oooooooooooo 0mm000mmmm0m0 GCUAAAUUCUGUG 353 13-13386 oCTGF-1042- 13388 Chl oooooooooooo mm000mmmm0m00 CUAAAUUCUGUGG 35413-13388 o CTGF-755- 13390 Chl oooooooooooo 000m0m0mmm00m AGACACGUUUGGC355 13-13390 o CTGF-467- 13392 Chl oooooooooooo mm0m0000mm00mCCGCAAGAUCGGC 356 13-13392 o CTGF-995- 13394 Chl oooooooooooom0mm000mmm000 UAUCAAGUUUGAG 357 13-13394 o CTGF-927- 13396 Chloooooooooooo 0000mm00mmm00 GAAGCUGACCUGG 358 13-13396 o SPP1-1025- 13398Chl oooooooooooo mmm0m000mm000 CUCAUGAAUUAGA 359 13-13398 o SPP1-1049-13400 Chl oooooooooooo mm0000mm00mm0 CUGAGGUCAAUUA 360 13-13400 oSPP1-1051- 13402 Chl oooooooooooo 0000mm00mm000 GAGGUCAAUUAAA 36113-13402 o SPP1-1048- 13404 Chl oooooooooooo mmm0000mm00mm UCUGAGGUCAAUU362 13-13404 o SPP1-1050- 13406 Chl oooooooooooo m0000mm00mm00UGAGGUCAAUUAA 363 13-13406 o SPP1-1047- 13408 Chl oooooooooooommmm0000mm00m UUCUGAGGUCAAU 364 13-13408 o SPP1-800- 13410 Chloooooooooooo 0mm00mm000m00 GUCAGCUGGAUGA 365 13-13410 o SPP1-492- 13412Chl oooooooooooo mmmm00m000mmm UUCUGAUGAAUCU 366 13-13412 o SPP1-612-13414 Chl oooooooooooo m000mm0000mm0 UGGACUGAGGUCA 367 13-13414 oSPP1-481- 13416 Chl oooooooooooo 000mmmm0mm0mm GAGUCUCACCAUU 36813-13416 o SPP1-614- 13418 Chl oooooooooooo 00mm0000mm000 GACUGAGGUCAAA369 13-13418 o SPP1-951- 13420 Chl oooooooooooo mm0m00mm0m000UCACAGCCAUGAA 370 13-13420 o SPP1-482- 13422 Chl oooooooooooo00mmmm0mm0mmm AGUCUCACCAUUC 371 13-13422 o SPP1-856- 13424 Chloooooooooooo 000m000000mm0 AAGCGGAAAGCCA 372 13-13424 o SPP1-857- 13426Chl oooooooooooo 00m000000mm00 AGCGGAAAGCCAA 373 13-13426 o SPP1-365-13428 Chl oooooooooooo 0mm0m0m000m00 ACCACAUGGAUGA 374 13-13428 oSPP1-359- 13430 Chl oooooooooooo 0mm0m00mm0m0m GCCAUGACCACAU 37513-13430 o SPP1-357- 13432 Chl oooooooooooo 000mm0m00mm0m AAGCCAUGACCAC376 13-13432 o SPP1-858- 13434 Chl oooooooooooo 0m000000mm00mGCGGAAAGCCAAU 377 13-13434 o SPP1-1012- 13436 Chl oooooooooooo000mmmm0m0mmm AAAUUUCGUAUUU 378 13-13436 o SPP1-1014- 13438 Chloooooooooooo 0mmmm0m0mmmmm AUUUCGUAUUUCU 379 13-13438 o SPP1-356- 13440Chl oooooooooooo 0000mm0m00mm0 AAAGCCAUGACCA 380 13-13440 o SPP1-368-13442 Chl oooooooooooo 0m0m000m00m0m ACAUGGAUGAUAU 381 13-13442 oSPP1-1011- 13444 Chl oooooooooooo 0000mmmm0m0mm GAAAUUUCGUAUU 38213-13444 o SPP1-754- 13446 Chl oooooooooooo 0m0mmmmmm00mm GCGCCUUCUGAUU383 13-13446 o SPP1-1021- 13448 Chl oooooooooooo 0mmmmmm0m000mAUUUCUCAUGAAU 384 13-13448 o SPP1-1330- 13450 Chl oooooooooooommmmm0m000m00 CUCUCAUGAAUAG 385 13-13450 o SPP1-346- 13452 Chloooooooooooo 000mmm00m0000 AAGUCCAACGAAA 386 13-13452 o SPP1-869- 13454Chl oooooooooooo 0m00m00000m00 AUGAUGAGAGCAA 387 13-13454 o SPP1-701-13456 Chl oooooooooooo 0m000000mm000 GCGAGGAGUUGAA 388 13-13456 oSPP1-896- 13458 Chl oooooooooooo m00mm00m00mm0 UGAUUGAUAGUCA 38913-13458 o SPP1-1035- 13460 Chl oooooooooooo 000m00m0m0mmm AGAUAGUGCAUCU390 13-13460 o SPP1-1170- 13462 Chl oooooooooooo 0m0m0m0mmm0mmAUGUGUAUCUAUU 391 13-13462 o SPP1-1282- 13464 Chl oooooooooooommmm0m0000000 UUCUAUAGAAGAA 392 13-13464 o SPP1-1537- 13466 Chloooooooooooo mm0mmm00m00mm UUGUCCAGCAAUU 393 13-13466 o SPP1-692- 13468Chl oooooooooooo 0m0m000000m00 ACAUGGAAAGCGA 394 13-13468 o SPP1-840-13470 Chl oooooooooooo 0m00mmm000mm0 GCAGUCCAGAUUA 395 13-13470 oSPP1-1163- 13472 Chl oooooooooooo m00mm000m0m0m UGGUUGAAUGUGU 39613-13472 o SPP1-789- 13474 Chl oooooooooooo mm0m0000m000m UUAUGAAACGAGU397 13-13474 o SPP1-841- 13476 Chl oooooooooooo m00mmm000mm0mCAGUCCAGAUUAU 398 13-13476 o SPP1-852- 13478 Chl oooooooooooo0m0m000m00000 AUAUAAGCGGAAA 399 13-13478 o SPP1-209- 13480 Chloooooooooooo m0mm00mm000m0 UACCAGUUAAACA 400 13-13480 o SPP1-1276- 13482Chl oooooooooooo m0mmm0mmmm0m0 UGUUCAUUCUAUA 401 13-13482 o SPP1-137-13484 Chl oooooooooooo mm00mm0000000 CCGACCAAGGAAA 402 13-13484 oSPP1-711- 13486 Chl oooooooooooo 000m00m0m0m0m GAAUGGUGCAUAC 40313-13486 o SPP1-582- 13488 Chl oooooooooooo 0m0m00m00mm00 AUAUGAUGGCCGA404 13-13488 o SPP1-839- 13490 Chl oooooooooooo 00m00mmm000mmAGCAGUCCAGAUU 405 13-13490 o SPP1-1091- 13492 Chl oooooooooooo0m0mmm00mm000 GCAUUUAGUCAAA 406 13-13492 o SPP1-884- 13494 Chloooooooooooo 00m0mmmm00m0m AGCAUUCCGAUGU 407 13-13494 o SPP1-903- 13496Chl oooooooooooo m00mm00000mmm UAGUCAGGAACUU 408 13-13496 o SPP1-1090-13498 Chl oooooooooooo m0m0mmm00mm00 UGCAUUUAGUCAA 409 13-13498 oSPP1-474- 13500 Chl oooooooooooo 0mmm00m000mmm GUCUGAUGAGUCU 41013-13500 o SPP1-575- 13502 Chl oooooooooooo m000m0m0m0m00 UAGACACAUAUGA411 13-13502 o SPP1-671- 13504 Chl oooooooooooo m000m00000m0mCAGACGAGGACAU 412 13-13504 o SPP1-924- 13506 Chl oooooooooooom00mm0m000mmm CAGCCGUGAAUUC 413 13-13506 o SPP1-1185- 13508 Chloooooooooooo 00mmm00000m00 AGUCUGGAAAUAA 414 13-13508 o SPP1-1221- 13510Chl oooooooooooo 00mmm0m00mmmm AGUUUGUGGCUUC 415 13-13510 o SPP1-347-13512 Chl oooooooooooo 00mmm00m00000 AGUCCAACGAAAG 416 13-13512 oSPP1-634- 13514 Chl oooooooooooo 000mmmm0m000m AAGUUUCGCAGAC 41713-13514 o SPP1-877- 13516 Chl oooooooooooo 00m00m000m0mm AGCAAUGAGCAUU418 13-13516 o SPP1-1033- 13518 Chl oooooooooooo mm000m00m0m0mUUAGAUAGUGCAU 419 13-13518 o SPP1-714- 13520 Chl oooooooooooom00m0m0m0m000 UGGUGCAUACAAG 420 13-13520 o SPP1-791- 13522 Chloooooooooooo 0m0000m000mm0 AUGAAACGAGUCA 421 13-13522 o SPP1-813- 13524Chl oooooooooooo mm0000m0mm000 CCAGAGUGCUGAA 422 13-13524 o SPP1-939-13526 Chl oooooooooooo m00mm0m000mmm CAGCCAUGAAUUU 423 13-13526 oSPP1-1161- 13528 Chl oooooooooooo 0mm00mm000m0m AUUGGUUGAAUGU 42413-13528 o SPP1-1164- 13530 Chl oooooooooooo 00mm000m0m0m0 GGUUGAAUGUGUA425 13-13530 o SPP1-1190- 13532 Chl oooooooooooo 00000m00mm00mGGAAAUAACUAAU 426 13-13532 o SPP1-1333- 13534 Chl oooooooooooomm0m000m00000 UCAUGAAUAGAAA 427 13-13534 o SPP1-537- 13536 Chloooooooooooo 0mm00m00mm000 GCCAGCAACCGAA 428 13-13536 o SPP1-684- 13538Chl oooooooooooo m0mmmm0m0m0m0 CACCUCACACAUG 429 13-13538 o SPP1-707-13540 Chl oooooooooooo 00mm000m00m0m AGUUGAAUGGUGC 430 13-13540 oSPP1-799- 13542 Chl oooooooooooo 00mm00mm000m0 AGUCAGCUGGAUG 43113-13542 o SPP1-853- 13544 Chl oooooooooooo m0m000m000000 UAUAAGCGGAAAG432 13-13544 o SPP1-888- 13546 Chl oooooooooooo mmmm00m0m00mmUUCCGAUGUGAUU 433 13-13546 o SPP1-1194- 13548 Chl oooooooooooo0m00mm00m0m0m AUAACUAAUGUGU 434 13-13548 o SPP1-1279- 13550 Chloooooooooooo mm0mmmm0m0000 UCAUUCUAUAGAA 435 13-13550 o SPP1-1300- 13552Chl oooooooooooo 00mm0mm0mm0m0 AACUAUCACUGUA 436 13-13552 o SPP1-1510-13554 Chl oooooooooooo 0mm00mm0mmm0m GUCAAUUGCUUAU 437 13-13554 oSPP1-1543- 13556 Chl oooooooooooo 00m00mm00m000 AGCAAUUAAUAAA 43813-13556 o SPP1-434- 13558 Chl oooooooooooo 0m00mmmm00m00 ACGACUCUGAUGA439 13-13558 o SPP1-600- 13560 Chl oooooooooooo m00m0m00mmm0mUAGUGUGGUUUAU 440 13-13560 o SPP1-863- 13562 Chl oooooooooooo000mm00m00m00 AAGCCAAUGAUGA 441 13-13562 o SPP1-902- 13564 Chloooooooooooo 0m00mm00000mm AUAGUCAGGAACU 442 13-13564 o SPP1-921- 13566Chl oooooooooooo 00mm00mm0m000 AGUCAGCCGUGAA 443 13-13566 o SPP1-154-13568 Chl oooooooooooo 0mm0mm0m00000 ACUACCAUGAGAA 444 13-13568 oSPP1-217- 13570 Chl oooooooooooo 000m000mm00mm AAACAGGCUGAUU 44513-13570 o SPP1-816- 13572 Chl oooooooooooo 000mmm0000mm GAGUGCUGAAACC446 13-13572 o SPP1-882- 13574 Chl oooooooooooo m000m0mmmm00mUGAGCAUUCCGAU 447 13-13574 o SPP1-932- 13576 Chl oooooooooooo00mmmm0m00mm0 AAUUCCACAGCCA 448 13-13576 o SPP1-1509- 13578 Chloooooooooooo m0mm00mm0mmm0 UGUCAAUUGCUUA 449 13-13578 o SPP1-157- 13580Chl oooooooooooo 0mm0m00000mm0 ACCAUGAGAAUUG 450 13-13580 o SPP1-350-13582 Chl oooooooooooo mm00m00000mm0 CCAACGAAAGCCA 451 13-13582 oSPP1-511- 13584 Chl oooooooooooo mm00mm0mm00mm CUGGUCACUGAUU 45213-13584 o SPP1-605- 13586 Chl oooooooooooo m00mmm0m000mm UGGUUUAUGGACU453 13-13586 o SPP1-811- 13588 Chl oooooooooooo 00mm0000m0mm0GACCAGAGUGCUG 454 13-13588 o SPP1-892- 13590 Chl oooooooooooo00m0m00mm00m0 GAUGUGAUUGAUA 455 13-13590 o SPP1-922- 13592 Chloooooooooooo 0mm00mm0m000m GUCAGCCGUGAAU 456 13-13592 o SPP1-1169- 13594Chl oooooooooooo 00m0m0m0mmm0m AAUGUGUAUCUAU 457 13-13594 o SPP1-1182-13596 Chl oooooooooooo mm000mmm00000 UUGAGUCUGGAAA 458 13-13596 oSPP1-1539- 13598 Chl oooooooooooo 0mmm00m00mm00 GUCCAGCAAUUAA 45913-13598 o SPP1-1541- 13600 Chl oooooooooooo mm00m00mm00m0 CCAGCAAUUAAUA460 13-13600 o SPP1-427- 13602 Chl oooooooooooo 00mmm000m00mmGACUCGAACGACU 461 13-13602 o SPP1-533- 13604 Chl oooooooooooo0mmm0mm00m00m ACCUGCCAGCAAC 462 13-13604 o APOB--13- 13763 Chloooooooooooo 0m+00+m0+m0+m ACtGAaUAcCAaU 463 13763 TEG o APOB--13- 13764Chl oooooooooooo 0mm000m0mm00m ACUGAAUACCAAU 464 13764 TEG o MAP4K4--16-13766 Chl oooooooooooo DY547mm0m0000 CUGUGGAAGUCUA 465 13766 o 0mmm0PPIB--13- 13767 Chl oooooooooooo mmmmmmmmmmmmm GGCUACAAAAACA 466 13767 oPPIB--15- 13768 Chl oooooooooooo mm00mm0m00000 UUGGCUACAAAAA 467 13768ooo m0 CA PPIB--17- 13769 Chl oooooooooooo 0mmm00mm0m000 AUUUGGCUACAAA468 13769 ooooo 00m0 AACA MAP4K4--16- 13939 Chl oooooooooooom0m0000m0mmm0 UGUAGGAUGUCUA 469 13939 o APOB-4314- 13940 Chloooooooooooo 0mmm0000000m0 AUCUGGAGAAACA 470 16-13940 o APOB-4314- 13941Chl oooooooooooo 000mmm0000000 AGAUCUGGAGAAA 471 17-13941 ooo m0 CAAPOB--16- 13942 Chl oooooooooooo 00mmm0mmm0mm0 GACUCAUCUGCUA 472 13942 oAPOB--18- 13943 Chl oooooooooooo 00mmm0mmm0mm0 GACUCAUCUGCUA 473 13943 oAPOB--17- 13944 Chl oooooooooooo m000mmm0mmm0m UGGACUCAUCUGC 474 13944ooo m0 UA APOB--19- 13945 Chl oooooooooooo m000mmm0mmm0m UGGACUCAUCUGC475 13945 ooo m0 UA APOB-4314- 13946 Chl oooooooooooo 0000000m00m0mGGAGAAACAACAU 476 16-13946 o APOB-4314- 13947 Chl oooooooooooomm0000000m00m CUGGAGAAACAAC 477 17-13947 ooo 0m AU APOB--16- 13948 Chloooooooooooo 00mmmmmm000m0 AGUCCCUCAAACA 478 13948 o APOB--17- 13949 Chloooooooooooo 0000mmmmmm000 AGAGUCCCUCAAA 479 13949 ooo m0 CA APOB--16-13950 Chl oooooooooooo 0mm000m0mm00m ACUGAAUACCAAU 480 13950 o APOB--18-13951 Chl oooooooooooo 0mm000m0mm00m ACUGAAUACCAAU 481 13951 o APOB--17-13952 Chl oooooooooooo 0m0mm000m0mm0 ACACUGAAUACCA 482 13952 ooo 0m AUAPOB--19- 13953 Chl oooooooooooo 0m0mm000m0mm0 ACACUGAAUACCA 483 13953ooo 0m AU MAP4K4--16- 13766.2 Chl oooooooooooo DY547mm0m0000CUGUGGAAGUCUA 484 13766.2 o 0mmm0 CTGF-1222- 13980 Chl oooooooooooo0m0000000m0m0 ACAGGAAGAUGUA 485 16-13980 o CTGF-813- 13981 Chloooooooooooo 000m0000mmmm GAGUGGAGCGCCU 486 16-13981 o CTGF-747- 13982Chl oooooooooooo m0mm000000m0 CGACUGGAAGACA 487 16-13982 o CTGF-817-13983 Chl oooooooooooo 0000mmmm0mmm GGAGCGCCUGUUC 488 16-13983 oCTGF-1174- 13984 Chl oooooooooooo 0mm0mm0m00mm0 GCCAUUACAACUG 48916-13984 o CTGF-1005- 13985 Chl oooooooooooo 000mmmmmm00mm GAGCUUUCUGGCU490 16-13985 o CTGF-814- 13986 Chl oooooooooooo 00m0000mmmm0AGUGGAGCGCCUG 491 16-13986 o CTGF-816- 13987 Chl oooooooooooom0000mmmm0mm UGGAGCGCCUGUU 492 16-13987 o CTGF-1001- 13988 Chloooooooooooo 0mmm000mmmmmm GUUUGAGCUUUCU 493 16-13988 o CTGF-1173- 13989Chl oooooooooooo m0mm0mm0m00mm UGCCAUUACAACU 494 16-13989 o CTGF-749-13990 Chl oooooooooooo 0mm000000m0m ACUGGAAGACACG 495 16-13990 oCTGF-792- 13991 Chl oooooooooooo 00mm0mmm00mmm AACUGCCUGGUCC 49616-13991 o CTGF-1162- 13992 Chl oooooooooooo 000mmm0m0mmm0 AGACCUGUGCCUG497 16-13992 o CTGF-811- 13993 Chl oooooooooooo m0000m0000mmCAGAGUGGAGCGC 498 16-13993 o CTGF-797- 13994 Chl oooooooooooommm00mmm000mm CCUGGUCCAGACC 499 16-13994 o CTGF-1175- 13995 Chloooooooooooo mm0mm0m00mm0m CCAUUACAACUGU 500 16-13995 o CTGF-1172- 13996Chl oooooooooooo mm0mm0mm0m00m CUGCCAUUACAAC 501 16-13996 o CTGF-1177-13997 Chl oooooooooooo 0mm0m00mm0mmm AUUACAACUGUCC 502 16-13997 oCTGF-1176- 13998 Chl oooooooooooo m0mm0m00mm0mm CAUUACAACUGUC 50316-13998 o CTGF-812- 13999 Chl oooooooooooo 0000m0000mmm AGAGUGGAGCGCC504 16-13999 o CTGF-745- 14000 Chl oooooooooooo 0mm0mm000000ACCGACUGGAAGA 505 16-14000 o CTGF-1230- 14001 Chl oooooooooooo0m0m0m0000m0 AUGUACGGAGACA 506 16-14001 o CTGF-920- 14002 Chloooooooooooo 0mmmm0m000mm GCCUUGCGAAGCU 507 16-14002 o CTGF-679- 14003Chl oooooooooooo 0mm0m00000m0 GCUGCGAGGAGUG 508 16-14003 o CTGF-992-14004 Chl oooooooooooo 0mmm0mm000mmm GCCUAUCAAGUUU 509 16-14004 oCTGF-1045- 14005 Chl oooooooooooo 00mmmm0m0000m AAUUCUGUGGAGU 51016-14005 o CTGF-1231- 14006 Chl oooooooooooo m0m0m0000m0m UGUACGGAGACAU511 16-14006 o CTGF-991- 14007 Chl oooooooooooo 00mmm0mm000mmAGCCUAUCAAGUU 512 16-14007 o CTGF-998- 14008 Chl oooooooooooom000mmm000mmm CAAGUUUGAGCUU 513 16-14008 o CTGF-1049- 14009 Chloooooooooooo mm0m0000m0m0m CUGUGGAGUAUGU 514 16-14009 o CTGF-1044- 14010Chl oooooooooooo 000mmmm0m0000 AAAUUCUGUGGAG 515 16-14010 o CTGF-1327-14011 Chl oooooooooooo mmmm00m00m0m0 UUUCAGUAGCACA 516 16-14011 oCTGF-1196- 14012 Chl oooooooooooo m00m00m0mmmmm CAAUGACAUCUUU 51716-14012 o CTGF-562- 14013 Chl oooooooooooo 00m0mm00m0m0m AGUACCAGUGCAC518 16-14013 o CTGF-752- 14014 Chl oooooooooooo 000000m0mmmmGGAAGACACGUUU 519 16-14014 o CTGF-994- 14015 Chl oooooooooooomm0mm000mmm00 CUAUCAAGUUUGA 520 16-14015 o CTGF-1040- 14016 Chloooooooooooo 00mm000mmmm0m AGCUAAAUUCUGU 521 16-14016 o CTGF-1984- 14017Chl oooooooooooo 000m0000m0m00 AGGUAGAAUGUAA 522 16-14017 o CTGF-2195-14018 Chl oooooooooooo 00mm00mm00mmm AGCUGAUCAGUUU 523 16-14018 oCTGF-2043- 14019 Chl oooooooooooo mmmm0mmm000m0 UUCUGCUCAGAUA 52416-14019 o CTGF-1892- 14020 Chl oooooooooooo mm0mmm000mm00 UUAUCUAAGUUAA525 16-14020 o CTGF-1567- 14021 Chl oooooooooooo m0m0m00m00m0UAUACGAGUAAUA 526 16-14021 o CTGF-1780- 14022 Chl oooooooooooo00mm000m00mmm GACUGGACAGCUU 527 16-14022 o CTGF-2162- 14023 Chloooooooooooo 0m00mmmmm0mm0 AUGGCCUUUAUUA 528 16-14023 o CTGF-1034- 14024Chl oooooooooooo 0m0mm00mm000 AUACCGAGCUAAA 529 16-14024 o CTGF-2264-14025 Chl oooooooooooo mm0mm00000m0m UUGUUGAGAGUGU 530 16-14025 oCTGF-1032- 14026 Chl oooooooooooo 0m0m0mm00mm0 ACAUACCGAGCUA 53116-14026 o CTGF-1535- 14027 Chl oooooooooooo 00m0000000mm0 AGCAGAAAGGUUA532 16-14027 o CTGF-1694- 14028 Chl oooooooooooo 00mm0mmmmmm00AGUUGUUCCUUAA 533 16-14028 o CTGF-1588- 14029 Chl oooooooooooo0mmm0000m0m00 AUUUGAAGUGUAA 534 16-14029 o CTGF-928- 14030 Chloooooooooooo 000mm00mmm000 AAGCUGACCUGGA 535 16-14030 o CTGF-1133- 14031Chl oooooooooooo 00mm0m0000000 GGUCAUGAAGAAG 536 16-14031 o CTGF-912-14032 Chl oooooooooooo 0m00mm000mmmm AUGGUCAGGCCUU 537 16-14032 oCTGF-753- 14033 Chl oooooooooooo 00000m0mmmm0 GAAGACACGUUUG 538 16-14033o CTGF-918- 14034 Chl oooooooooooo 000mmmm0m000 AGGCCUUGCGAAG 53916-14034 o CTGF-744- 14035 Chl oooooooooooo m0mm0mm00000 UACCGACUGGAAG540 16-14035 o CTGF-466- 14036 Chl oooooooooooo 0mmm0000mm0ACCGCAAGAUCGG 541 16-14036 o CTGF-917- 14037 Chl oooooooooooom000mmmm0m00 CAGGCCUUGCGAA 542 16-14037 o CTGF-1038- 14038 Chloooooooooooo m00mm000mmmm CGAGCUAAAUUCU 543 16-14038 o CTGF-1048- 14039Chl oooooooooooo mmm0m0000m0m0 UCUGUGGAGUAUG 544 16-14039 o CTGF-1235-14040 Chl oooooooooooo m0000m0m00m0 CGGAGACAUGGCA 545 16-14040 oCTGF-868- 14041 Chl oooooooooooo 0m00m00mmmmm AUGACAACGCCUC 546 16-14041o CTGF-1131- 14042 Chl oooooooooooo 0000mm0m00000 GAGGUCAUGAAGA 54716-14042 o CTGF-1043- 14043 Chl oooooooooooo m000mmmm0m000 UAAAUUCUGUGGA548 16-14043 o CTGF-751- 14044 Chl oooooooooooo m000000m0mmmUGGAAGACACGUU 549 16-14044 o CTGF-1227- 14045 Chl oooooooooooo0000m0m0m000 AAGAUGUACGGAG 550 16-14045 o CTGF-867- 14046 Chloooooooooooo 00m00m00mmmm AAUGACAACGCCU 551 16-14046 o CTGF-1128- 14047Chl oooooooooooo 00m000mm0m00 GGCGAGGUCAUGA 552 16-14047 o CTGF-756-14048 Chl oooooooooooo 00m0m0mmm00mm GACACGUUUGGCC 553 16-14048 oCTGF-1234- 14049 Chl oooooooooooo 0m00000m0m00m ACGGAGACAUGGC 55416-14049 o CTGF-916- 14050 Chl oooooooooooo mm000mmmm0m00 UCAGGCCUUGCGA555 16-14050 o CTGF-925- 14051 Chl oooooooooooo 0m0000mm00mmmGCGAAGCUGACCU 556 16-14051 o CTGF-1225- 14052 Chl oooooooooooo000000m0m0m00 GGAAGAUGUACGG 557 16-14052 o CTGF-445- 14053 Chloooooooooooo 0m00mmmm00mmm GUGACUUCGGCUC 558 16-14053 o CTGF-446- 14054Chl oooooooooooo m00mmmm00mmmm UGACUUCGGCUCC 559 16-14054 o CTGF-913-14055 Chl oooooooooooo m00mm000mmmm0 UGGUCAGGCCUUG 560 16-14055 oCTGF-997- 14056 Chl oooooooooooo mm000mmm000mm UCAAGUUUGAGCU 56116-14056 o CTGF-277- 14057 Chl oooooooooooo 0mm0000mm0m00 GCCAGAACUGCAG562 16-14057 o CTGF-1052- 14058 Chl oooooooooooo m0000m0m0mOmmUGGAGUAUGUACC 563 16-14058 o CTGF-887- 14059 Chl oooooooooooo0mm0000000m00 GCUAGAGAAGCAG 564 16-14059 o CTGF-914- 14060 Chloooooooooooo 00mm000mmmm0m GGUCAGGCCUUGC 565 16-14060 o CTGF-1039- 14061Chl oooooooooooo 000mm000mmmm0 GAGCUAAAUUCUG 566 16-14061 o CTGF-754-14062 Chl oooooooooooo 000m0m0mmm00 AAGACACGUUUGG 567 16-14062 oCTGF-1130- 14063 Chl oooooooooooo m0000mm0m0000 CGAGGUCAUGAAG 56816-14063 o CTGF-919- 14064 Chl oooooooooooo 00mmmm0m0000m GGCCUUGCGAAGC569 16-14064 o CTGF-922- 14065 Chl oooooooooooo mmm0m0000mm00CUUGCGAAGCUGA 570 16-14065 o CTGF-746- 14066 Chl oooooooooooomm00mm000000m CCGACUGGAAGAC 571 16-14066 o CTGF-993- 14067 Chloooooooooooo mmm0mm000mmm0 CCUAUCAAGUUUG 572 16-14067 o CTGF-825- 14068Chl oooooooooooo m0mmmm0000mmm UGUUCCAAGACCU 573 16-14068 o CTGF-926-14069 Chl oooooooooooo m0000mm00mmm0 CGAAGCUGACCUG 574 16-14069 oCTGF-923- 14070 Chl oooooooooooo mm0m0000mm00m UUGCGAAGCUGAC 57516-14070 o CTGF-866- 14071 Chl oooooooooooo m00m00m00m0mm CAAUGACAACGCC576 16-14071 o CTGF-563- 14072 Chl oooooooooooo 0m0mm00m0m0m0GUACCAGUGCACG 577 16-14072 o CTGF-823- 14073 Chl oooooooooooommm0mmmm0000m CCUGUUCCAAGAC 578 16-14073 o CTGF-1233- 14074 Chloooooooooooo m0m00000m0m00 UACGGAGACAUGG 579 16-14074 o CTGF-924- 14075Chl oooooooooooo m0m0000mm00mm UGCGAAGCUGACC 580 16-14075 o CTGF-921-14076 Chl oooooooooooo mmmm0m0000mm0 CCUUGCGAAGCUG 581 16-14076 oCTGF-443- 14077 Chl oooooooooooo mm0m00mmmm00m CUGUGACUUCGGC 58216-14077 o CTGF-1041- 14078 Chl oooooooooooo 0mm000mmmm0m0 GCUAAAUUCUGUG583 16-14078 o CTGF-1042- 14079 Chl oooooooooooo mm000mmmm0m00CUAAAUUCUGUGG 584 16-14079 o CTGF-755- 14080 Chl oooooooooooo000m0m0mmm00m AGACACGUUUGGC 585 16-14080 o CTGF-467- 14081 Chloooooooooooo mm0m0000mm00m CCGCAAGAUCGGC 586 16-14081 o CTGF-995- 14082Chl oooooooooooo m0mm000mmm000 UAUCAAGUUUGAG 587 16-14082 o CTGF-927-14083 Chl oooooooooooo 0000mm00mmm00 GAAGCUGACCUGG 588 16-14083 oSPP1-1091- 14131 Chl oooooooooooo 0m0mmm00mm000 GCAUUUAGUCAAA 58916-14131 o PPIB--16- 14188 Chl oooooooooooo mmmmmmmmmmmmm GGCUACAAAAACA590 14188 o PPIB--17- 14189 Chl oooooooooooo mm00mm0m00000 UUGGCUACAAAAA591 14189 ooo m0 CA PPIB--18- 14190 Chl oooooooooooo 0mmm00mm0m000AUUUGGCUACAAA 592 14190 ooooo 00m0 AACA pGL3-1172- 14386 chloooooooooooo 0m000m0m00mmm ACAAAUACGAUUU 593 16-14386 o pGL3-1172- 14387chl oooooooooooo DY5470m000m0m ACAAAUACGAUUU 594 16-14387 o 00mmmMAP4K4- 14390 Chl oooooooooooo Pmmmmmmmmmmmm CUUUGAAGAGUUC 595 2931-25-oooooooooooo 000mmmmmmmmmm UGUGGAAGUCUA 14390 o miR-122-- 14391 Chlssoooooooooo mmmmmmmmmmmmm ACAAACACCAUUG 596 23-14391 ooooooossssmmmmmmmmmm UCACACUCCA 14084 Chl oooooooooooo mmm0m000mm000 CUCAUGAAUUAGA719 o 14085 Chl oooooooooooo mm0000mm00mm0 CUGAGGUCAAUUA 720 o 14086 Chloooooooooooo 0000mm00mm000 GAGGUCAAUUAAA 721 o 14087 Chl oooooooooooommm0000mm00mm UCUGAGGUCAAUU 722 o 14088 Chl oooooooooooo m0000mm00mm00UGAGGUCAAUUAA 723 o 14089 Chl oooooooooooo mmmm0000mm00m UUCUGAGGUCAAU724 o 14090 Chl oooooooooooo 0mm00mm000m00 GUCAGCUGGAUGA 725 o 14091 Chloooooooooooo mmmm00m000mmm UUCUGAUGAAUCU 726 o 14092 Chl oooooooooooom000mm0000mm0 UGGACUGAGGUCA 727 o 14093 Chl oooooooooooo 000mmmm0mm0mmGAGUCUCACCAUU 728 o 14094 Chl oooooooooooo 00mm0000mm000 GACUGAGGUCAAA729 o 14095 Chl oooooooooooo mm0m00mm0m000 UCACAGCCAUGAA 730 o 14096 Chloooooooooooo 00mmmm0mm0mmm AGUCUCACCAUUC 731 o 14097 Chl oooooooooooo000m00000mm0 AAGCGGAAAGCCA 732 o 14098 Chl oooooooooooo 00m00000mm00AGCGGAAAGCCAA 733 o 14099 Chl oooooooooooo 0mm0m0m000m00 ACCACAUGGAUGA734 o 14100 Chl oooooooooooo 0mm0m00mm0m0m GCCAUGACCACAU 735 o 14101 Chloooooooooooo 000mm0m00mm0m AAGCCAUGACCAC 736 o 14102 Chl oooooooooooo0m00000mm00m GCGGAAAGCCAAU 737 o 14103 Chl oooooooooooo 000mmmmm0mmmAAAUUUCGUAUUU 738 o 14104 Chl oooooooooooo 0mmmmm0mmmmm AUUUCGUAUUUCU739 o 14105 Chl oooooooooooo 0000mm0m00mm0 AAAGCCAUGACCA 740 o 14106 Chloooooooooooo 0m0m000m00m0m ACAUGGAUGAUAU 741 o 14107 Chl oooooooooooo0000mmmmm0mm GAAAUUUCGUAUU 742 o 14108 Chl oooooooooooo 0mmmmmmm00mmGCGCCUUCUGAUU 743 o 14109 Chl oooooooooooo 0mmmmmm0m000m AUUUCUCAUGAAU744 o 14110 Chl oooooooooooo mmmmm0m000m00 CUCUCAUGAAUAG 745 o 14111 Chloooooooooooo 000mmm00m000 AAGUCCAACGAAA 746 o 14112 Chl oooooooooooo0m00m00000m00 AUGAUGAGAGCAA 747 o 14113 Chl oooooooooooo 0m00000mm000GCGAGGAGUUGAA 748 o 14114 Chl oooooooooooo m00mm00m0Omm0 UGAUUGAUAGUCA749 o 14115 Chl oooooooooooo 000m00m0m0mmm AGAUAGUGCAUCU 750 o 14116 Chloooooooooooo 0m0m0m0mmm0mm AUGUGUAUCUAUU 751 o 14117 Chl oooooooooooommmm0m0000000 UUCUAUAGAAGAA 752 o 14118 Chl oooooooooooo mm0mmm00m00mmUUGUCCAGCAAUU 753 o 14119 Chl oooooooooooo 0m0m000000m0 ACAUGGAAAGCGA754 o 14120 Chl oooooooooooo 0m00mmm000mm0 GCAGUCCAGAUUA 755 o 14121 Chloooooooooooo m00mm000m0m0m UGGUUGAAUGUGU 756 o 14122 Chl oooooooooooomm0m0000m00m UUAUGAAACGAGU 757 o 14123 Chl oooooooooooo m00mmm000mm0mCAGUCCAGAUUAU 758 o 14124 Chl oooooooooooo 0m0m000m0000 AUAUAAGCGGAAA759 o 14125 Chl oooooooooooo m0mm00mm000m0 UACCAGUUAAACA 760 o 14126 Chloooooooooooo m0mmm0mmmm0m0 UGUUCAUUCUAUA 761 o 14127 Chl oooooooooooomm0mm0000000 CCGACCAAGGAAA 762 o 14128 Chl oooooooooooo 000m00m0m0m0mGAAUGGUGCAUAC 763 o 14129 Chl oooooooooooo 0m0m00m00mm0 AUAUGAUGGCCGA764 o 14130 Chl oooooooooooo 00m00mmm000mm AGCAGUCCAGAUU 765 o 14132 Chloooooooooooo 00m0mmmm0m0m AGCAUUCCGAUGU 766 o 14133 Chl oooooooooooom00mm00000mmm UAGUCAGGAACUU 767 o 14134 Chl oooooooooooo m0m0mmm00mm00UGCAUUUAGUCAA 768 o 14135 Chl oooooooooooo 0mmm00m000mmm GUCUGAUGAGUCU769 o 14136 Chl oooooooooooo m000m0m0m0m00 UAGACACAUAUGA 770 o 14137 Chloooooooooooo m000m0000m0m CAGACGAGGACAU 771 o 14138 Chl oooooooooooom00mmm000mmm CAGCCGUGAAUUC 772 o 14139 Chl oooooooooooo 00mmm00000m00AGUCUGGAAAUAA 773 o 14140 Chl oooooooooooo 00mmm0m00mmmm AGUUUGUGGCUUC774 o 14141 Chl oooooooooooo 00mmm00m0000 AGUCCAACGAAAG 775 o 14142 Chloooooooooooo 000mmmmm000m AAGUUUCGCAGAC 776 o 14143 Chl oooooooooooo00m00m000m0mm AGCAAUGAGCAUU 777 o 14144 Chl oooooooooooo mm000m00m0m0mUUAGAUAGUGCAU 778 o 14145 Chl oooooooooooo m00m0m0m0m000 UGGUGCAUACAAG779 o 14146 Chl oooooooooooo 0m0000m00mm0 AUGAAACGAGUCA 780 o 14147 Chloooooooooooo mm0000m0mm000 CCAGAGUGCUGAA 781 o 14148 Chl oooooooooooom00mm0m000mmm CAGCCAUGAAUUU 782 o 14149 Chl oooooooooooo 0mm00mm000m0mAUUGGUUGAAUGU 783 o 14150 Chl oooooooooooo 00mm000m0m0m0 GGUUGAAUGUGUA784 o 14151 Chl oooooooooooo 00000m00mm00m GGAAAUAACUAAU 785 o 14152 Chloooooooooooo mm0m000m00000 UCAUGAAUAGAAA 786 o 14153 Chl oooooooooooo0mm00m00mm00 GCCAGCAACCGAA 787 o 14154 Chl oooooooooooo m0mmmm0m0m0m0CACCUCACACAUG 788 o 14155 Chl oooooooooooo 00mm000m00m0m AGUUGAAUGGUGC789 o 14156 Chl oooooooooooo 00mm00mm000m0 AGUCAGCUGGAUG 790 o 14157 Chloooooooooooo m0m000m00000 UAUAAGCGGAAAG 791 o 14158 Chl oooooooooooommmm0m0m00mm UUCCGAUGUGAUU 792 o 14159 Chl oooooooooooo 0m00mm00m0m0mAUAACUAAUGUGU 793 o 14160 Chl oooooooooooo mm0mmmm0m0000 UCAUUCUAUAGAA794 o 14161 Chl oooooooooooo 00mm0mm0mm0m0 AACUAUCACUGUA 795 o 14162 Chloooooooooooo 0mm00mm0mmm0m GUCAAUUGCUUAU 796 o 14163 Chl oooooooooooo00m00mm00m000 AGCAAUUAAUAAA 797 o 14164 Chl oooooooooooo 0m0mmmm00m00ACGACUCUGAUGA 798 o 14165 Chl oooooooooooo m00m0m00mmm0m UAGUGUGGUUUAU799 o 14166 Chl oooooooooooo 000mm00m00m00 AAGCCAAUGAUGA 800 o 14167 Chloooooooooooo 0m00mm00000mm AUAGUCAGGAACU 801 o 14168 Chl oooooooooooo00mm00mmm000 AGUCAGCCGUGAA 802 o 14169 Chl oooooooooooo 0mm0mm0m00000ACUACCAUGAGAA 803 o 14170 Chl oooooooooooo 000m000mm00mm AAACAGGCUGAUU804 o 14171 Chl oooooooooooo 000m0mm0000mm GAGUGCUGAAACC 805 o 14172 Chloooooooooooo m000m0mmmm0m UGAGCAUUCCGAU 806 o 14173 Chl oooooooooooo00mmmm0m00mm0 AAUUCCACAGCCA 807 o 14174 Chl oooooooooooo m0mm00mm0mmm0UGUCAAUUGCUUA 808 o 14175 Chl oooooooooooo 0mm0m00000mm0 ACCAUGAGAAUUG809 o 14176 Chl oooooooooooo mm00m0000mm0 CCAACGAAAGCCA 810 o 14177 Chloooooooooooo mm00mm0mm00mm CUGGUCACUGAUU 811 o 14178 Chl oooooooooooom00mmm0m000mm UGGUUUAUGGACU 812 o 14179 Chl oooooooooooo 00mm0000m0mm0GACCAGAGUGCUG 813 o 14180 Chl oooooooooooo 00m0m00mm00m0 GAUGUGAUUGAUA814 o 14181 Chl oooooooooooo 0mm00mmm000m GUCAGCCGUGAAU 815 o 14182 Chloooooooooooo 00m0m0m0mmm0m AAUGUGUAUCUAU 816 o 14183 Chl oooooooooooomm000mmm00000 UUGAGUCUGGAAA 817 o 14184 Chl oooooooooooo 0mmm00m00mm00GUCCAGCAAUUAA 818 o 14185 Chl oooooooooooo mm00m00mm00m0 CCAGCAAUUAAUA819 o 14186 Chl oooooooooooo 00mmm00m0mm GACUCGAACGACU 820 o 14187 Chloooooooooooo 0mmm0mm00m00m ACCUGCCAGCAAC 821 o

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety. This applicationincorporates by reference the entire contents, including all thedrawings and all parts of the specification (including sequence listingor amino acid/polynucleotide sequences) of the co-pending U.S.Provisional Application No. 61/135,855, filed on Jul. 24, 2008, entitled“SHORT HAIRPIN RNAI CONSTRUCTS AND USES THEREOF,” and U.S. ProvisionalApplication No. 61/197,768, filed on Oct. 30, 2008, entitled “MINIRNACONSTRUCTS AND USES THEREOF.”

What is claimed is: 1-91. (canceled)
 92. An isolated double strandednucleic acid molecule comprising a guide strand and a passenger strand,wherein the isolated double stranded nucleic acid molecule includes adouble stranded region and a single stranded region, wherein the doublestranded region is from 8-15 nucleotides long, wherein the singlestranded region is at the 3′ end of the guide strand and is 4-12nucleotides long, wherein the single stranded region contains 3, 4, 5,6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, wherein atleast 40% of the nucleotides of the isolated double stranded nucleicacid molecule are modified, and wherein the isolated double strandednucleic acid molecule does not form a hairpin.
 93. The isolated doublestranded nucleic acid molecule of claim 92, wherein the double strandedregion is 11, 12, 13, or 14 nucleotides long and/or wherein the singlestranded region is at least 6 or at least 7 nucleotides long.
 94. Theisolated double stranded nucleic acid molecule of claim 92, wherein eachnucleotide within the single stranded region has a phosphorothioatemodification.
 95. The isolated double stranded nucleic acid molecule ofclaim 92, wherein at least one of the nucleotides of the isolated doublestranded nucleic acid molecule that is modified comprises a 2′ O-methylor a 2′-fluoro modification and/or wherein at least one of thenucleotides of the isolated double stranded nucleic acid molecule thatis modified comprises a hydrophobic modification.
 96. The isolateddouble stranded nucleic acid molecule of claim 92, wherein the guidestrand of the double stranded nucleic acid molecule exhibitscomplementarity to a gene encoding for Osteopontin (SPP1), SOD1 orMAP4K4, optionally wherein the guide strand comprises SEQ ID NO:170, SEQID NO:40 or SEQ ID NO:25.
 97. An isolated asymmetric nucleic acidmolecule comprising: a first polynucleotide wherein the firstpolynucleotide is complementary to a second polynucleotide and a targetgene; and a second polynucleotide, wherein the second polynucleotide isat least 6 nucleotides shorter than the first polynucleotide, whereinthe first polynucleotide includes a single stranded region of 6, 7, 8,9, 10, 11 or 12 nucleotides, wherein the single stranded region of thefirst polynucleotide contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12phosphorothioate modifications, wherein the asymmetric nucleic acidmolecule also includes a double stranded region of 8-15 nucleotideslong, and wherein at least 50% of C and U nucleotides in the doublestranded region are 2′ O-methyl modified or 2′-fluoro modified.
 98. Theisolated asymmetric nucleic acid molecule of claim 97, wherein thesingle stranded region is 6 or 7 nucleotides long and/or wherein eachnucleotide within the single stranded region has a phosphorothioatemodification.
 99. An isolated double stranded nucleic acid moleculecomprising: a guide strand of 17-21 nucleotides in length that hascomplementarity to a target gene, and a passenger strand of 8-16nucleotides in length, wherein the isolated double stranded nucleic acidmolecule includes a double stranded region of 8-15 nucleotides long anda single stranded region, wherein the guide strand and the passengerstrand form the double stranded nucleic acid molecule having the doublestranded region and the single stranded region, wherein the singlestranded region is at the 3′ end of the guide strand and is 4-12nucleotides in length, wherein the single stranded region comprises 2-12phosphorothioate modifications, wherein at least 40% of the nucleotidesof the isolated double stranded nucleic acid molecule are modified, andwherein the isolated double stranded nucleic acid molecule does not forma hairpin.
 100. The isolated double stranded nucleic acid molecule ofclaim 99, wherein the isolated double stranded nucleic acid moleculecontains at least one hydrophobic base modification and wherein thehydrophobic base modification comprises a hydrophobic modification of apyrimidine base, optionally at position 4 or 5, optionally wherein thehydrophobic base modification is selected from the group consisting of aphenyl, 4-pyridyl, 2-pyridyl, indolyl, isobutyl, tryptophanyl(C₈H₆N)CH₂CH(NH₂)CO), methyl, butyl, aminobenzyl, and naphthylmodification of a uridine or cytidine.
 101. A method for inhibiting theexpression of a target gene in a mammalian cell, comprising contactingthe mammalian cell with an isolated double stranded nucleic acidmolecule comprising a guide strand and a passenger strand, wherein theisolated double stranded nucleic acid molecule includes a doublestranded region and a single stranded region, wherein the doublestranded is from 8-15 nucleotides long, wherein the single strandedregion is at the 3′ end of the guide and is 4-12 nucleotides long,wherein the single stranded region of the guide strand contains 3, 4, 5,6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, wherein atleast 40% of the nucleotides of the isolated double stranded nucleicacid molecule are modified, and wherein the isolated double strandednucleic acid molecule does not form a hairpin.
 102. The method of claim101, wherein the double stranded is 11, 12, 13, or 14 nucleotides longand/or wherein the single stranded region is at least 6 or at least 7nucleotides long.
 103. The method of claim 101, wherein each nucleotidewithin the single stranded region has a phosphorothioate modification.104. The method of claim 101, wherein at least one of the nucleotides ofthe isolated double stranded nucleic acid molecule that is modifiedcomprises a 2′ O-methyl or a 2′-fluoro modification and/or wherein atleast one of the nucleotides of the isolated double stranded nucleicacid molecule that is modified comprises a hydrophobic modification.105. The method of claim 101, wherein the double stranded nucleic acidmolecule exhibits complementarity to a gene encoding for Osteopontin(SPP1), SOD1 or MAP4K4, optionally wherein the guide strand comprisesSEQ ID NO:170, SEQ ID NO:40 or SEQ ID NO:25.
 106. An isolated doublestranded nucleic acid molecule comprising: a double stranded nucleicacid molecule non-covalently complexed to a hydrophobic molecule,wherein the hydrophobic molecule is a polycationic molecule.
 107. Theisolated double stranded nucleic acid molecule of claim 106, wherein thepolycationic molecule is selected from the group consisting ofprotamine, arginine rich peptides, and spermine.
 108. An isolated doublestranded nucleic acid molecule comprising: a double stranded nucleicacid molecule, wherein the double stranded nucleic acid molecule isdouble stranded RNA, directly complexed to a hydrophobic moleculewithout a linker, wherein the hydrophobic molecule is not cholesterol.109. A composition comprising: a double stranded nucleic acid molecule,wherein the double stranded nucleic acid molecule is double strandedRNA, attached to a hydrophobic molecule, wherein the double strandednucleic acid molecule comprises a guide strand and a passenger strand,wherein the guide strand is from 16-29 nucleotides long and issubstantially complementary to a target gene, wherein the passengerstrand is from 8-14 nucleotides long and has complementarity to theguide strand, wherein position 1 of the guide strand is 5′phosphorylated or has a 2′ O-methyl modification, wherein at least 40%of the nucleotides of the double stranded nucleic acid are modified, andwherein the double stranded nucleic acid molecule has one end that isblunt or includes a one-two nucleotide overhang; a neutral fattymixture; and optionally a cargo molecule, wherein the double strandednucleic acid molecule and the neutral fatty mixture forms a micelle.110. The composition of claim 109, wherein the composition is sterile.111. The composition of claim 109, wherein the neutral fatty mixturecomprises a DOPC (dioleoylphosphatidylcholine).
 112. The composition ofclaim 109, wherein the neutral fatty mixture comprises a DSPC(distearoylphosphatidylcholine).
 113. The composition of claim 109,wherein the neutral fatty mixture further comprises a sterol.
 114. Thecomposition of claim 113, wherein the sterol is cholesterol.
 115. Thecomposition of claim 109, wherein the composition includes at least 20%DOPC and at least 20% cholesterol.
 116. The composition of claim 109,wherein the cargo molecule is a fusogenic lipid and preferably is atleast 10% fusogenic lipid.
 117. The composition of claim 116, whereinthe fusogenic lipid is DOPE.